WO2015000856A1 - Lignée de cellules chorioniques humaines immortalisées ainsi que procédé pour immortaliser des cellules chorioniques humaines - Google Patents

Lignée de cellules chorioniques humaines immortalisées ainsi que procédé pour immortaliser des cellules chorioniques humaines Download PDF

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WO2015000856A1
WO2015000856A1 PCT/EP2014/063866 EP2014063866W WO2015000856A1 WO 2015000856 A1 WO2015000856 A1 WO 2015000856A1 EP 2014063866 W EP2014063866 W EP 2014063866W WO 2015000856 A1 WO2015000856 A1 WO 2015000856A1
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cell line
genes
cells
chorionic
gene
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Günter CICHON
Jonas Stanke
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Charité – Universitätsmedizin Berlin
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Charité – Universitätsmedizin Berlin
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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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • the invention relates to an immortalized chorion cell line and a method for producing immortal cell lines from human embryonic chorionic tissue, and to the use of the cell line for producing recombinant protein and / or medicinal active substances.
  • tumor cells are used only in exceptional cases, as these are burdened by the presence of some undefined oncogenes or viral pathogens.
  • cell lines of animal origin e.g., COS cells
  • COS cells are more likely to be used to produce recombinant proteins.
  • the use of animal cell lines is burdened by undesirable contamination of the product with animal proteins, so that cell lines, which are of human origin but have no malignant background, have special value for the production of medicinal agents.
  • ESchG Embryo Protection Act
  • the invention is based on the finding that embryonic cells have many advantages as starting points for the production of cell lines.
  • EP 1 161 548 B2 describes the production of recombinant proteins in human cells by using adenovirus E1 protein coding sequences.
  • EP 1 161 548 B2 discloses a method for producing at least one recombinant protein in a cell comprising providing a cell comprising in its genome sequences encoding E1A and E1B of an adenovirus, the cell being in its genome does not include a sequence encoding a structural adenoviral protein, and wherein the cell is of a human embryonic
  • Retinoblast is derived.
  • Retinoblasts are the precursor cells of the retina. Their extraction is problematic, because it is essential to directly access to vital cells, which can be obtained only in the context of a termination of pregnancy.
  • An immortalized cell line is disclosed, for example, in WO 2001/036615 A2.
  • the cell line disclosed therein relates to a permanent amniocytic cell line containing at least one nucleic acid which effects the expression of the gene products of the E1 region of adenovirus.
  • the use of chorionic tissue as a starting point for the production of an immortalized cell line is neither disclosed nor suggested in WO 2001/036615 A2.
  • Chorionic tissue using adenoviral genes has hitherto not been disclosed or suggested in the prior art.
  • the invention therefore has the aim to use a different starting material, which is easy to win and beyond advantageous for further use.
  • the starting material used is preferably the embryonic portion of the placenta (the embryonic chorion).
  • the invention is therefore based on the object of providing means and corresponding methods by which substances can be synthesized which can ultimately serve as medicaments. This problem is solved by the features of claim 1. Further advantageous
  • the invention therefore relates to an immortalized human chorionic cell line.
  • the embryonic part of the placenta consists of the chorion plate, which rests on a thin egg skin (the amnion).
  • the maternal part of the placenta is the so-called decidua.
  • the chorion plate represents the main tissue component of the embryonic placenta and bears the brunt of the
  • Chorionic cells naturally synthesize a large number of endocrine factors and proteins, making them particularly suitable for use as a producer cell line for expression of recombinant human proteins and vaccines (MHF Sullivan, Endocrine cell lines from the placenta, Molecular and Cellular Endocrinology 228 (2004) 103 -1 19,
  • chorionic tissue not described. Immortalization of chorionic tissue using adenoviral genes has the advantage that the oncogenes used (adenovirus E1 genes) in the context of human cells only have a comparatively low transformation potential. In humans, no malignant tumors are known which are triggered by adenovirus E1 genes, so that the production of medical devices (recombinant proteins, vaccines, growth factors, hormones, etc.) on cell lines immortalized by adenovirus E1 genes appears to be safer from a biological safety point of view.
  • the invention also relates to an immortalized human chorionic cell originally derived from the embryonic portion of the placenta (the embryonic chorion).
  • the terms "cell line” and "cell” are both to be used for a description of the present invention.
  • Cell lines are cells of a tissue type that can breed in the course of cell culture. Both immortalized (immortal) cell lines and primary cells cultured (primary culture) are known in the art.
  • a primary culture is a non-immortalized cell culture obtained directly from a tissue.
  • the invention therefore relates to immortalized (immortal) cell lines and cells comprising
  • Chorionic tissue or chorionic cells are generated.
  • Immortalization is the removal of the limit on the number of
  • immortalized cells can divide as often as desired and can be propagated essentially indefinitely in cell culture.
  • a "permanent" or “immortalized” cell line is meant, according to the present invention, that the corresponding cells are in some way genetically engineered to grow substantially permanently in cell culture.
  • the immortalized cell line of the present invention enables the production of recombinant cells Proteins, recombinant adenoviruses or other medicinal agents in a cell line without risk of undesired contamination of the recombinant product with safety-relevant oncogenes
  • the immortalized human chorionic cell lines of the present invention are very well suited by their biological properties for the production of recombinant drugs.
  • the cell line (or the individual cells of the cell line) comprises nucleic acid sequences (or at least one nucleic acid) which encode one or more E1 genes of an adenovirus and preferably cause expression of the E1 gene products.
  • the E1 genes the E1a, E1b1 and / or E1b2 genes are preferred.
  • the (at least one) nucleic acid or nucleic acid sequences which causes the expression of the E1 gene products of adenovirus may be any suitable nucleic acid or nucleic acids which result in stable expression of these gene products.
  • the nucleic acid sequences are preferably exogenous nucleic acid sequences. Exogenous nucleic acid sequences are understood as meaning all sequences which have been used as transgene or other nucleic acid newly introduced into the cell (after isolation of the cell). To achieve the desired immortalization of cells over extended periods of time
  • the E1 sequences can be detected by PCR amplification from the genome of
  • Adenovirus type 5 complete genome: http://www.ncbi.nlm.nih.goV/nuccore/AC_000008.1).
  • E1 genes of human adenovirus serotype 5 were chosen for immortalization. In principle immortalization is also possible with E1 genes other adenoviruses of the 57 previously known different adenovirus serotypes.
  • Immortalization plasmids used Ad5 base position 437-3366: Preferred nucleotide sequence of the adenoviral E1 genes (SEQ ID NO: 1):
  • the aforementioned preferred adenoviral gene sequences encode the following
  • Ad5 E1a SEQ ID NO: 2
  • Ad5-E1b (19K) (SEQ ID NO: 3):
  • Ad5-E1b (55K) (SEQ ID NO: 4):
  • extrachromosomal e.g. as an episomally replicating plasmid or mini-chromosome.
  • extrachromosomal e.g. as an episomally replicating plasmid or mini-chromosome.
  • the expression of the various gene products can be one and the same
  • Nucleic acid molecule or, for example, different nucleic acid molecules are effected.
  • expression is understood in the prior art, the process of producing the product of a gene that is either a specific protein that causes a specific feature or a specific property, or RNA forms that are not translated into proteins (eg antisense RNAs, tRNAs).
  • RNA forms that are not translated into proteins (eg antisense RNAs, tRNAs).
  • suitable options for achieving the desired expression will be apparent to the person skilled in the art.
  • the introduction of an adenoviral E1 gene that partially deregulates the cell cycle shows a surprisingly good rate of transformation without the need for tumor-inducing agents.
  • adenoviral E1 genes to immortalize chorionic cells provides a means of generating a chorionic cell line by low-risk and non-tumor-inducing agents. Since the immortalized cell lines produced on the basis of the adenoviral E1 genes are non-tumorigenic, the recombinant cells produced in this cell line are Proteins for medical use in humans are significantly safer than proteins produced in an immortalized cancer cell line.
  • Another embodiment of the invention relates to immortalized human chorionic cells comprising nucleic acid sequences that result in (preferred continuous) expression of the CAR receptor.
  • the CAR receptor (cosackie and adenovirus receptor) improves the infectivity of chorionic cells with recombinant adenoviruses, which is advantageous in terms of the production of recombinant proteins and the production of adenoviral vaccines.
  • a particular aspect of the invention relates to the manner of linking the genes of the CAR receptor and the E1 genes required for the immortalization.
  • the linkage of these two genes is preferably in a manner that allows long term stable expression of the CAR receptor, although the CAR receptor does not contribute to the immortalization of the chorionic cells.
  • telomere is the reverse transcriptase subunit of the human telomerase complex (hTERT).
  • telomere sequences are deposited in online genebanks (e.g., NCBI; http://www.ncbi.nlm.nih.goV/nuccore/AB085628.1).
  • Preferred nucleotide sequence human telomerase (SEQ ID NO. 5):
  • the (at least one) nucleic acid or nucleic acid sequences which brings about the expression of the telomerase can be any suitable nucleic acid or nucleic acids which lead to a stable expression of these gene products.
  • Nucleic acid sequences are preferably exogenous nucleic acid sequences. Exogenous nucleic acid sequences are understood as meaning all sequences which have been used as transgene or other nucleic acid newly introduced into the cell (after isolation of the cell).
  • the invention implements the artificial expression of key proteins useful for the
  • Immortality of the cells are effective, for example, the telomerase, the one
  • telomeres Preventing chromosome end-disruption during DNA replication in eukaryotes.
  • Telomerase restores the ends of the chromosomes, called telomeres. Immortalization by telomerase also provides a way to immortalize the cells by non-tumorigenic transformation.
  • telomere By using telomerase and / or adenoviral E1 genes, the telomerase and / or adenoviral E1 genes, the telomerase and / or adenoviral E1 genes, the telomerase and / or adenoviral E1 genes, the telomerase and / or adenoviral E1 genes, the telomerase and / or adenoviral E1 genes.
  • chorion cells are immortalized without significant phenotypic changes in native cells.
  • Another embodiment of the invention relates to an immortalized human
  • Chorion cell line characterized in that the nucleic acid sequences encoding one or more E1 genes of an adenovirus and / or telomerase are integrated in the genome at the locus of a tumor suppressor gene (e.g., p53 or Rb).
  • a tumor suppressor gene e.g., p53 or Rb.
  • Tumor suppressor gene refers to all genomic regions associated with the expression of the tumor suppressor gene.
  • such regions are the tumor suppressor encoding gene, the promoter of the gene and / or others
  • Another aspect of the invention relates to a method for producing an immortal chorionic cell line, wherein human embryonic chorionic tissue is used as a starting tissue, which is immortalized by means of adenoviral E1 genes and / or human telomorase.
  • a further subject of the present invention is therefore a process for producing a permanent chorion cell line, in particular one as defined above
  • Chorion cell line characterized by transfecting chorionic cells with at least one nucleic acid which causes expression of the adenoviral gene products of the E1 a region and / or E1 b region, or a telomerase.
  • the resulting cell clones may then optionally be further isolated and cloned to obtain single cell lines, if desired.
  • the invention relates to a method for producing an immortal chorion cell line, comprising the following steps: a) providing and / or obtaining chorion cells, preferably of human
  • embryonic chorionic tissue as starting material; comprehensively favoring the culturing of the cells; b) transfecting the cells with nucleic acid sequences (or at least one
  • Nucleic acid encoding one or more E1 genes of an adenovirus and preferentially effecting the expression of the E1 gene products, and / or transfecting the cells with nucleic acid sequences (or at least one nucleic acid) encoding human telomerase and preferentially effecting the expression of telomerase; c) culturing the cells in cell culture; d) subcloning of the cells, whereby the cells are diluted so that (preferably under microscopic control) cell populations emerge, which emerge from a single cell. e) Cultivation of the cells in cell culture.
  • provision and / or recovery means any process that is suitable for the preparation of chorionic tissue.
  • Chorionic tissue obtained by biopsy. Alternatively, it can also be obtained from tissue obtained as part of spontaneous abortion and human genetic
  • the inventive method can also be carried out with already recovered or isolated chorionic tissue.
  • Biopsy extraction of chorionic tissue is also known as chorionic biopsy (chorion biopsy).
  • the invention therefore relates in a preferred embodiment to a method in which human embryos are not destroyed.
  • embryonic cells which are preferably obtained in the context of human genetic diagnostics and are usually discarded after completion of the studies, or cells obtained by biopsy can be used.
  • the invention may be characterized, in association with one or more of the embodiments disclosed herein, as a method of immortalizing human chorionic cells or as a method of producing immortal chorionic cell lines.
  • the method of the invention may comprise separating the chorionic cells from chorionic villus biopsy material.
  • the chorionic cells can be separated from amniocytes to produce the desired ones
  • Isolate chorionic cells as starting material.
  • Isolate chorionic cells as starting material.
  • a preferred embodiment (preferably under sterile conditions)
  • Chorionic tissue used in culture medium and then treated with an enzymatic digestion may comprise a trypsin and / or collagenase treatment, preferably for at least 10 minutes, or between 10 to 120 minutes, preferably between 20 and 60 minutes.
  • recombinant enzymes of non-animal origin are used here to reduce the risk of
  • the cells growing in cell culture are transfected with the nucleic acids according to the invention.
  • the isolated or provided chorionic cells can be cultured only about 4-6 passages. After successful integration and continuous expression of adenoviral E1 genes or telomerase, the growth inhibition is abolished and the relative proportion of transfected cells in the cell culture increases continuously.
  • the proportion of E1-expressing cells is about 15 to 35%, preferably 25%.
  • the proportion of human serum in the culture medium is maintained at 20% to 40%, preferably 30%.
  • the cells are subcloned.
  • the cells are isolated, for example under microscopic control in 96-well plates, so that it is ensured that a newly growing cell population from a single
  • the proportion of human serum in the culture medium is reduced to 15 to 20%, and after further 2 to 4, preferably 3 weeks to 7 to 15, preferably 10%. Subsequently, the proportion of human serum in the culture medium should be reduced 2 to 4 weeks later, preferably 3 weeks later to 2.5 to 7%, preferably 5%, then 2 to 4 weeks, preferably 3 weeks later to 1% to 5% , preferably 2.5%, and finally reduced to 0%. From this point on, the cells are preferably cultured under serum-free conditions (eg in 293-SFM (life technologies)).
  • the rate of division of the fastest clones is preferably 24-36 hours and the immortalization is complete.
  • transfection or “transfection” is understood here to mean any process which is suitable for introducing said nucleic acid (s) into the cells, examples of which are electroporation, liposomal systems of any type and combinations of these processes. Coprecipitation may be used In this type of transfection, the DNA is introduced into a mixture of calcium chloride and sodium phosphate.
  • the at least one nucleic acid which effects the expression of the adenoviral E1 gene products or telomerase may preferably be genomic DNA, cDNA, synthetic DNA, RNA and mRNA.
  • the nucleic acid is used in the form of a DNA expression vector. Examples include integrative vectors, bacterial plasmids, episomally replicating plasmids or mini-chromosomes. Preference is given to expression plasmids obtained by transfection into the genome of the recipient cell for
  • At least one nucleic acid is meant that the expression-causing elements can be located on one and the same nucleic acid as well as on different nucleic acids. For example, separate nucleic acids may be provided for expression of the gene products of the E1A and E1B regions.
  • the method according to the invention is characterized
  • the gene for the CAR receptor is expressed.
  • the invention further relates to a method for producing an immortal
  • Chorionic cell lines in which the gene used for immortalization e.g., adenovirus E1 genes or telomerase
  • shuts down by targeted homologous recombination to the locus of a tumor suppressor gene e.g., p53 or Rb
  • a tumor suppressor gene e.g., p53 or Rb
  • the invention relates to a process for the production of an immortal chorionic cell line in which the expression-controlling elements of the tumor suppressor gene are preserved, so that the loss of the
  • Tumorsuppressorgens reflectively increased promoter performance for the expression of the transgene (eg adenoviral E1 genes or telomerase) is used.
  • the transgene eg adenoviral E1 genes or telomerase
  • the invention relates to a method for the production of immortal chorion cell lines, which is characterized in that the insertion of the transgene DNA sequences are used which are homologous to the
  • the invention therefore relates to an immortalized human chorionic cell line, which is obtained by the production method according to the invention.
  • the immortalized human chorionic cell line may optionally be characterized by one or more of the method features or corresponding structural features disclosed in connection with the method.
  • the invention further relates to the use of the immortalized human chorion cell line according to the invention for the production of recombinant proteins and / or
  • a method of producing recombinant proteins which is performed on the basis of the immortalized human chorionic cell line, the cells containing a suitable expression cassette encoding one or more E1 genes of an adenovirus and / or a telomerase.
  • the recombinant protein produced is a human protein.
  • any protein can be expressed as the recombinant protein.
  • medicinally relevant human proteins are preferred.
  • human recombinant proteins are Human Growth Hormone (rHGH), Human Insulin (BHI), Follicle Stimulating Hormone (FSH), Factor VIII Kogenate, Erythropoietin (EPO), Granulocyte Colony Stimulating Factor (G-CSF), Filgrastim, alpha - galactosidase A alpha-L-iduronidase (rhIDU; laronidase), N-acetyl-4-sulfatase (rhASB;
  • Galsulfase tissue plasminogen activator (TPA), glucocerebrosidase, interferon (IF) or interferon beta-1b, or insulin-like growth factor 1 (IGF-1).
  • TPA tissue plasminogen activator
  • IF interferon
  • IGF-1b insulin-like growth factor 1
  • the chorion cell line according to the invention is generally suitable in particular for the production of adenoviral vectors which are characterized by deletions of the E1A and E1B genes.
  • Another aspect of the invention relates to a method for the production of recombinant adenoviruses using the immortalized human chorionic cell lines of the invention.
  • the recombinant adenoviruses E1 / E3 are deleted adenoviruses.
  • the invention further relates to the use of genes of the adenoviral E1 region for immortalizing a chorionic cell, wherein the genes of the adenoviral E1 region a Sequence identity to so-called E1-deleted adenoviruses of less than 90%, preferably less than 70% sequence identity, more preferably less than 50% sequence identity, to reduce the risk of unwanted recombination between the E1 -deleased adenoviruses and genes of the adenoviral E1 region to minimize.
  • a preferred embodiment is characterized in that the genes of the adenoviral E1 region do not have sufficient sequence homologies to so-called E1 -deleted adenoviruses in order to allow recombination between the E1 -deleased adenoviruses and genes of the adenoviral E1 region.
  • sequence identity can be determined by routine measures. A person skilled in the field of molecular biology will be able to check whether a certain sequence identity reduces the probability of recombination.
  • nucleic acid sequences can be altered without the
  • Amino acid sequence of the encoded protein change. Degenerate codons and thus the redundancy of the genetic code can be used for this. This can minimize the risks of recombination without causing functional changes in the protein.
  • the invention further relates to a method in which adenoviral E1 genes are introduced by homologous recombination into the genome of the (chorion) target cell in a manner which simultaneously leads to inactivation of individual tumor suppressor genes and in this way in a shortened period of time and in one higher proportion contributes to the immortalization and transformation of target cells.
  • Immortalization uses gene (e.g., adenovirus E1 genes or telomerase) targeted by homologous recombination at the locus of a tumor suppressor gene (e.g., p53 or Rb) and turns it off by the integration event.
  • gene e.g., adenovirus E1 genes or telomerase
  • a tumor suppressor gene e.g., p53 or Rb
  • the Amnion (Amnios, also sheep skin) is the thin, vaseless innermost egg skin and thus part of the amniotic sac of the amniotes (reptiles, birds and mammals).
  • the chorion (Zottenhaut) forms the middle and the decidua (Siebhaut) the external Eihaut.
  • the chorion is the outer part of the two embryonic layers of the amniotic sac, while the decidua belongs to the maternal part of the amniotic sac.
  • the chorion consists of the extraembryonic mesoderm and the trophoblast and forms the pediatric or embryonic part of the placenta, while the amnion presents the innermost layered epithelium of the amniotic sac and is responsible for a significant proportion of the production of the amniotic fluid.
  • Amnion and chorion differ from their anatomical location, function and genetic expression profile.
  • the chorionic tissue represents the main part of the embyonal placental tissue and bears the brunt of the metabolism between mother and child. About the Choriongwebe the embryo is supplied with nutrients and growth factors.
  • choroid cells similar to the liver are capable of producing a large number of hormones (such as human chorionic gonadotropin (HCG), pregnancy-associated protein A (PAPP-A), human placental lactogen (hPL), placental corticotropin releasing hormone),
  • Leukocyte protease inhibitors Elafine
  • enzymes or coagulation modulating factors Factor VII activating protease, Factor XIII
  • Factor VII activating protease Factor XIII
  • Diagnostics also a different way of sampling (either by amniocentesis or chorionic villus sampling). Furthermore, chorionic tissue accumulates in the context of abortions. While the fetus is inevitably lost in the event of abortion, the chorionic tissue remains vital for a longer period of time, from which in turn the chorion cells can be obtained. Due to the different
  • amniocentesis amniotic fluid by syringe and centrifuged the ammnocytes floating in it. Chorionic cells are never recovered in this way because there are no chorionic cells in the amniotic fluid.
  • the shiny, child-facing side of the placenta is the thin skin (Amnion), which can be detached from the placenta by a simple pull. Underneath, the chorionic tissue is displayed.
  • amnion and chorionic cells have morphological similarities in cell culture, they differ in their expression profile.
  • the chorion forms the so-called trophoblasts and amniotic cells by the height and the expression profile of so-called trophoblast markers such as CDX2, alpha and ß-HCG.
  • trophoblast markers such as CDX2, alpha and ß-HCG.
  • EOMES, EpCam / TROP1, ERR beta / NR3B2, FGF-4 and TROP-2 (Plouzek et al Placenta 1993
  • Chorionic tissue is therefore a biological material that becomes available without having to kill embryos.
  • the chorionic villus sampling also known as chorion biopsy, which is frequently performed as part of a pregnancy diagnosis, makes such material available in principle and without any moral-ethical complications.
  • the shaggy chorionic tissue is the precursor of the mother cake. It contains - as a rule - the same genetic information as the body cells of the embryo. The cells are usually harvested between the 10th and 12th week of gestation. There are two possible ways to remove the chorionic villi: (a) Transabdominal Method: Sub
  • chorionic cells In order to obtain and isolate endogenous proteins and active substances naturally produced by chorionic cells, as well as for use as a production platform for recombinant proteins (antibodies, coagulation factors, vaccines, hormones, etc.), chorionic cells are particularly suited to other embryonic tissues, since the production of growth hormones and nutrients belongs to their physiological determination.
  • a surprising aspect of the invention consists in the use of chorionic tissue for the establishment of new human cell lines, which, like amniotic cells, are uncritically accessible in the context of human genetic diagnostics, but not only by another anatomical situation but also by a different functional background distinguish different expression profile of amniotic cells.
  • a chorionic villi biopsy can usually be carried out earlier than an amniocentesis (since a certain amount of amniotic fluid must be present for the amniocentesis).
  • amniocentesis since a certain amount of amniotic fluid must be present for the amniocentesis.
  • early embryonic development chorionic tissue has an increased susceptibility to neoplastic developmental disorders (bladder moles, choriocarcinoma)
  • Chromosomal maldistribution indicating an increased tendency to transform and facilitates the attempt of intentional transformation or immortalization (eg by the stable expression of adenoviral E1 genes). Due to its physiological importance in the nutritive supply of the embryo and its diverse
  • chorionic tissues are particularly suitable for the establishment of cell lines for the production of recombinant drugs and proteins.
  • the immortalization or transformation of chorionic cells by stable transfection of one or more harmless to humans transgenes allows access to new human cell lines, which are characterized by a high biological safety profile and in a special way for the production of recombinant drugs are suitable.
  • Adenoviruses are a relatively homogeneous group of viruses characterized by an icosahedral capsid consisting mainly of the virally-encoded hexon, penton and fiber proteins and a linear, double-stranded DNA genome about 36 kilobases in size ( kb).
  • the viral genome contains at the ends the inverted terminal repeat sequences (ITRs), which are the viral origin of replication. Further, at the left end of the genome is the packaging signal required to package the viral genome into the viral capsids during an infection cycle.
  • ITRs inverted terminal repeat sequences
  • Adenoviruses have been isolated from many species.
  • Adenoviral vectors have mostly been derived from serotypes 2 (Ad2) and 5 (Ad5). Infections by Ad2 and Ad5 are endemic in humans. Ad2 and Ad5 are not oncogenic in humans and have a good one
  • E1 a is the first viral gene expressed after reaching the nucleus by the viral chromosome.
  • the E1 a gene encodes the 12S and HS proteins that are formed by alternative splicing of the E1 a RNA.
  • the E1A proteins activate the transcription of a number of cellular and viral genes through interaction with transcription factors.
  • the main functions of E1 a are a) the activation of the other early viral functions El b, E2, E3 and E4 and b) the induction of resting cells to enter the S phase of the cell cycle.
  • the sole expression of E1 A leads to programmed cell death of cells (apoptosis).
  • E1 B is one of the early viral genes activated by E1a.
  • the E1b gene encodes the EIB 55 kD protein and the EIB 19 kD protein, which result from alternative splicing of the E1b RNA.
  • the 55 kD protein modulates the progression of the cell cycle through interaction with the p53 tumor suppressor gene, is involved in the prevention of the p53 tumor suppressor gene
  • the E1b-19 kD protein is also important for the prevention of EIA-induced apoptosis of cells.
  • All human adenoviruses are capable of transforming cells in cell culture.
  • the co-expression of E1a and El b is generally preferred.
  • Embedded in the E1b transcription unit is the protein IX gene, which encodes a structural component of the viral capsid.
  • the genes of the so-called early region 1 (E1 region) of adenovirus serotype 5 (E1a, E1b1, E1b2) are not tumorogens for humans.
  • the cancer-inducing potential is other viral genes, e.g. that of HPV oncogenes E6 and E7 or that of SV40 virus (SV40 T antigen) is beyond dispute.
  • the use of genes of the adenoviral E1 region is therefore one way cells too
  • cell lines which have been immortalized by stable transfection of adenoviral E1 genes are suitable for the production of recombinant so-called E1 / E3 deleted adenoviruses, since the genes or gene functions deleted in the viruses functionally complement in this case by the packaging cell can be.
  • First generation adenoviral vectors (Gilardi et al., FEBS Letters 267, 60-62, 1990; Stratford-Perricaudet et al., Hum. Gene Ther. 1, 241-256, 1990) are characterized by deletions of E1a and E1 b genes, and / or the E3 gene.
  • E1 have a and El b
  • E1a is required for the activation of viral genes and E1b for the accumulation of viral transcripts.
  • E3 is also deleted to increase the capacity for uptake of foreign DNA. E3 is dispensable for the production of adenoviruses in cell culture. The capacity for the
  • Second generation adenoviral vectors are characterized by deletions of E2 and / or E4 in addition to deletions of E1a and Elb (Engelhardt et al., Proc. Natl. Acad. Sci., USA 91, 6196-6200, 1994; Yang et al., Nature Genet., 7, 362-367, 1994; Gorziglia et al., J. Virol 70, 4173-4178, 1996; Krougliak and Graham, Hum. Gene Ther. 6, 1575-1586, 1995;
  • Second generation adenoviral vectors have been developed to further reduce the transcription of viral genes and the expression of viral proteins and thus further reduce the antiviral immune response. The capacity for uptake of foreign DNA is not significantly increased over first generation adenoviral vectors. Second generation adenovirus vectors are produced in cell lines that complement the respective defect (E2 and / or E4) in addition to E1a and Elb.
  • the capacity for uptake of foreign DNA is about 37 kb, since the vast majority of the adenoviral genome is removed.
  • Various systems have been described for the preparation of adenoviral vectors of high DNA capacity (Kochanek et al., Supra; Parks et al., Proc Natl Acad Sci., USA 93, 13565-13570, 1996; Hardy et al., J Viral 71, 1842-1849, 1997).
  • the advantage of these adenoviral vectors with high DNA capacity compared to adenoviral vectors of the first and second generation lies in the greater capacity for uptake of foreign DNA and in lower toxicity and immunogenicity (Schiedner et al., Nature Genet. 18, 180- 183, 1998; Morral et al., Hum. Gene Ther. 9, 2709-2716, 1998).
  • the E1 region is replaced by a therapeutic gene.
  • the production of this recombinant, a therapeutically valuable gene-bearing virus is e.g. by cotransfection of a so-called helper or transfer plasmid, in which the transgene is flanked on both sides by regions which are homologous to the virus genome.
  • the generation of a recombinant virus requires homologous recombination between the transfer plasmid and the viral genome. Both plasmids alone can not generate adenoviruses. However, if a co-transfection of a 293 cell with the two plasmids occurs by chance, genetic information can be exchanged
  • Concentrations of the virus for therapeutic trials will increase these adenoviruses that are not replicable in normal cells in the 293 helper cell line. These cells stably integrate the missing E1 region into their genome, provide the gene product for virus replication and thus allow amplification of the recombinant adenovirus. These adenoviruses can not replicate in any other cellular system.
  • Coagulation factors membrane proteins or cell cycle proteins.
  • Proteins that can be expressed from vectors produced in transformed chorionic cells are dystrophin (Hoffman et al., Cell 51, 919, 1987, Factor VIII (Wion et al., Nature 317, 726 1985, Cystic Fibrous Transmembrane Regulator Protein (CFTR) (Anderson et al., Science 251, 679, 1991, Omithine transcarbamylase (OTC) (Murakami et al., J. Biol. Chem., 263, 18437, 1988, Alphai-antitrypsin (Fagerhol et al., Hum Genet., Vol.
  • HSV herpes simplex virus
  • Vectors produced in transformed choriocells may contain, in addition to the therapeutic gene, any reporter gene to better track the expression of the vector.
  • reporter genes include e.g. the ⁇ -galactosidase gene (Fowler et al., Proc Natl Acad., USA 74, 1507, 1977).
  • Vectors that can be produced in transformed choriocells may contain more than a single gene. The maximum number of genes that can be produced in such vectors depends on the uptake capacity of the respective vector and on the gene size.
  • the cell line produced according to the invention is also suitable for the expression of
  • the cultivation and immortalization of the chorion cells takes place exclusively in the absence of materials of animal origin.
  • culture media which contain only human sera, or recombinant proteins or other components (growth factors, insulin, recombinant trypsin, etc.) of non-animal origin.
  • This application further describes a method for immortalization in which the transforming adenoviral E1 genes are introduced into the genome of the target cell by homologous recombination.
  • the homologous recombination is preferably carried out in the Location of tumor suppressor genes (especially in the loci of tumor suppressor genes p53 and retinoblastoma gene (Rb)).
  • Homologous recombination eliminates the function of these tumor suppressor genes. At the same time, recombination occurs in a manner that leaves intact the expression controlling elements of the tumor suppressor genes, that is, the promoter region above the start codon of the tumor suppressor gene and the polyadenylation region below the stop codon of the tumor suppressor gene.
  • Recombination can be used for the expression of immortalization genes (E1 genes, telomerase), which after the loss of the tumor suppressor gene reflectively increased promoter performance (the tumor suppressor gene).
  • E1 genes immortalization genes
  • telomerase telomerase
  • the integration of cells used for immortalization of cells takes place randomly in transcriptionally active of the genome of the target cell. From the experience in the production of knock-in or knock-out mice, in which certain gene functions are targeted to be switched off or on, it is known that the probability of a directed integration to a particular gene locus in the genome can be increased by the fact that the Gene is flanked by gene segments that are homologous to the target region in the genome. Typically, the homologous recombination then takes place in a manner in which the genes lying between the homologous elements are exchanged for the previously integrated in the genome shares.
  • the below Figure 4 explains the principle using the example of integration in the p53 locus. Basically, this principle can be applied to everyone
  • apoptosis inner cell death
  • Immortalization especially of primary cells of human origin, since stable integration of the immortalization gene (eg adenoviral E1 genes or human telomerase) simultaneously eliminates tumor suppressor function of the target cell (e.g., p53 or Rb). Basically, the probability of a homologous
  • Tumor suppressor functions increase the likelihood of successful cell transformation by a factor of 5-10 over simple untargeted stable integration.
  • Tumor suppressors are proteins that control the cell cycle. It is not so much the name of a protein function as a physiological definition. A Mutation or deletion of a tumor suppressor-encoding gene (tumor suppressor gene) increases the likelihood of cell division remaining. In this sense, tumor suppressor genes have a comparable effect on mutations such as proto-oncogenes. Examples of tumor suppressors are: p16: This protein family inhibits the CDKs and thus prevents a transfer from the G1 to the S-phase of the cell cycle. p27:
  • This protein controls the transition from G0 to the G1 phase of the cell cycle and prevents it, if it is increasingly formed.
  • Both virus-affected cells promote p27 formation via a tissue mediator (TGF-ß) as well as neighboring cells in the
  • Rb Rb normally encloses the transcriptional factor E2F, which is essential for the initiation of the S-phase. Phosphorylation gradually opens the envelope, leaving the transcription factor "released".
  • E2F transcriptional factor
  • the p53 This protein usually ensures that a cell divides only if its genome is intact. The protein p53 is measurable in many types of degenerate cells in an increased amount. It is also present in normally growing cells, but hardly or not at all in resting cells. In many tumor types, the gene coding for p53 has mutated. In some but not all tumors, the protein appears to act as a tumor suppressor. p53 plays a role in the regulation of the cell cycle, where it slows the activity of a number of genes. p53 is found in all vertebrates.
  • p53 is used as integration target to integrate the transgenes of the invention in the genome.
  • homologous recombination is known to one skilled in the art and refers to an exchange between two DNA sequence regions with sufficient sequence identity.
  • homologous can also be understood as having high sequence identity or similarity in the nucleotide sequence, so homologs can account for more than a 50%, 60%, 70%, and “homologous" double sequence DNA sequences %, 80%, or preferably more than 90% sequence identity to one another. The invention therefore encompasses the possible application of a
  • Sequence-specific recombination namely a targeted (ie not random) integration of DNA into a genome by recombination.
  • Recombination is widespread in genetic engineering and well known to one skilled in the art of molecular biology.
  • tools are available today that can be used to artificially produce recombinant DNA and introduce it into organisms.
  • DNA sequences can be cut with restriction enzymes on specific recognition sequences and recombined with ligases.
  • plasmids or viruses serve as vectors to transfer the recombinant DNA into the target organism.
  • Alternative to Conventional DNA cloning with restriction enzymes and ligases are also known, based on homologous recombination-based technologies, such as recombineering
  • the invention encompasses the use of nucleic acid sequences encoding one or more E1 genes of an adenovirus and / or telomerase.
  • the nucleic acid sequences are preferably selected from the group comprising: a) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO. 1 or SEQ ID NO. 5;
  • nucleic acid molecule which is complementary to a nucleotide sequence according to a
  • c) a nucleic acid molecule which hybridizes with a nucleotide sequence according to a) or b) under stringent conditions hybridization conditions are known to a person skilled in the art, and are described, for example, in Sambrook, Molecular Cloning, Ed.1-3, Cold Spring Harbor, NY;
  • nucleic acid molecule comprising a nucleotide sequence which has a sufficient
  • nucleic acid molecule according to a nucleotide sequence according to a) to e) which is modified by deletions, additions, substitutions, translocations, inversions and / or insertions and is functionally analogous to a nucleotide sequence according to a) to e).
  • nucleic acid sequence provided that has sufficient sequence identity to be functionally analogous to the nucleic acid sequence according to item a), b) and / or c). For the purposes of the invention is called to the said nucleotide sequences or with these
  • Sequence variant can safely and effectively cause the desired immortalization of chorionic cells.
  • Functionally analogous sequences within the meaning of the invention are all
  • the nucleic acid molecule has at least 60%, preferably 70%, preferably 80%, very particularly preferably 90%, sequence identity to a nucleic acid molecule according to point a), b) and / or c).
  • Coexpression of the CAR receptor Almost all known viruses use mammalian cell infection to assist in the production of surface receptors that are naturally expressed by these mammalian cells and that have certain physiological functions in the context of a healthy cell. As part of an infection, the viruses first bind to the extracellular parts of these mostly membrane-bound receptors and then arrive in the context of natural mechanisms
  • Endocytosis inside the cell.
  • Human adenoviruses often use the CAR receptor for infection.
  • the natural role of the CAR receptor is binding to and
  • the level of expression of the CAR receptor in different tissues can vary widely, and embryonic nerve cells have a high number of CAR receptors, whereas they are barely detectable in adult neurons (Bergelson et al., Science 275 (5304): 1320 -3).
  • the CAR receptor In contrast to the expression of E1 genes, which are essential for the establishment and maintenance of the transformed state of a cell, the CAR receptor has
  • the expression of the CAR receptor has been linked to the expression of the E1 genes in a manner which makes a loss of the CAR receptor largely impossible ,
  • the CAR receptor gene and the adenoviral E1 genes have been linked together in a manner that ensures that E1 genes and the CAR receptor are always expressed together. This goal is achieved by the following arrangement of genes and expression-controlling elements. Just below the promoter (homologous regions) is the gene for the CAR receptor, which is followed by the complete gene of the adenoviral E1 region. Between the two genes lies a so-called
  • interribosomal entry site IRES site. If this expression cassette is stably incorporated into the genome of a host cell, the expression of the genes is necessarily always together, since initially only a so-called. Primary transcript (only one mRNA) is made, the but can bind to the ribosome via both the 5 'untranslated portion of the mRNA and the IRES sequence, resulting in the production of two different proteins from only one mRNA strand. Instead of a so-called IRES site, it is possible to introduce into the same target so-called self-cleaving sequences which also lead to the expression of two different gene products (see FIG. 5).
  • a plasmid was first commissioned for synthesis (gene art) carrying 1000 base pairs of the upstream region and 1000 base pairs of the downstream region of the human p53 gene.
  • a gene fragment from the genome of the human adenovirus serotype 5 genome was placed between these two p53 flanking sequences.
  • the gene fragment comprises the base pairs 438-3510 of the adenoviral wild-type genome.
  • At the 5 'end of this sequence is a short portion of the E1 -A promoter (position 438-559) and from base 560 the coding for E1 genes shares.
  • an additional polyadenylation signal was used (poly-A from bovine growth hormone).
  • FIG. 6 shows the p53-Ad5-E1 (final) plasmid.
  • the plasmid was cut with the restriction enzyme EcoRI, whereby unwanted sequence portions (ampicillin-resistance and the attachment site for bacterial DNA polymerase, the so-called origin of reolication (ori)) fall out of the plasmid (see plasmid map; 6).
  • the two gene fragments were separated on an agarose gel (0.8%) and the desired 5321 base pair segment was excised and purified.
  • the introduction of the DNA on chorion cells was carried out by calcium phosphate coprecipitation.
  • the DNA is transformed into a mixture of calcium chloride and
  • the source of primary chorionic cells is excess chorionic tissue, which is obtained after chorionic villus sampling, as well as the tissue produced as part of a natural abortion and subsequent genetic diagnosis.
  • tissue is then subjected to enzymatic digestion with trypsin / EDTA (0.05%) in medium (DMEM), first for about 30 minutes, and then preferably after about 60 minutes of digestion with collagenase.
  • trypsin / EDTA 0.05%) in medium (DMEM)
  • human recombinant trypsin from E. coli, biozol
  • recombinant collagen from Clostridium perfrigens, Sigma-Aldrich
  • the speed of the enzymatic digestion should be controlled microscopically every 10 minutes, since a too long incubation reduces the viability of the cells and too short digestion reduces the yield and only one
  • the process of cell release can be supported.
  • the cells released in this way are washed twice and cultured in DMEM culture medium (without animal additives) but with the addition of 30% human serum. After 24 hours, a change of medium and the dead cells are separated, since they can affect the growth of adhering to the bottom of the culture dish cells.
  • the cells are initially cultivated in 6-well plates (9 cm 2 per well). After the chorion cells have grown approximately 70% confluent, 10 ⁇ g pre-cut DNA (after EcoRI digestion) is evenly dripped into the cell medium by calcium transfection (in 200 ⁇ l transfection buffer). The size of the calcium crystals can be controlled under the microscope (the smaller the better). After 6 hours and after 24 hours, the medium is changed and the cells are washed once each gently with medium. If the transfection mixture is left on the cells for too long, too much of the cells will die off.
  • the proportion of E1-expressing cells is about 25%.
  • the proportion of human serum is kept at 30%. Now there is a subcloning of the cells.
  • the cells in a 96-well plate are thinned so strongly that under microscopic control in individual wells only a cell is detectable.
  • cell culture supernatant from the bulk culture is added for further cultivation (which still contains stimulating growth factors). Under these conditions, a complete tissue combination can be used again from individual cells.
  • the proportion of human serum is reduced to 20% every 3 weeks and then to 10%, then to 5%, to 2.5% and finally to 0% after a further 3 weeks. From this point on the cells are cultured under serum-free conditions in 293-SFM (life technologies).
  • FIG. 1 Organization of the adenovirus genome, location of the early genes.
  • the figure shows a scheme of organization of an adenovirus genome.
  • the approximately 35000 base pair genome is divided into 100 so-called mapunits (mu).
  • 1 mu measures approximately 350 base pairs (bp).
  • the genes of the so-called E1 region start at about 1.5 mu and end at about 10 mu, so span (involving the promoter and polyA region a range of about 3500 bp. Since the presence of these genes to a accelerated
  • Figure 4 Scheme of homologous recombination: The p53 gene is exchanged for the E1 gene without losing the expression-controlling elements of the p53 locus. In this way, after the loss of the p53 gene reflectively increased p53 promoter power can be used for expression of the transgene.
  • FIG. 5 The use of an interribosomal entry site (IRES) sequence between CAR receptor gene and E1 gene allows CAR receptor expression to be compulsorily linked to that of the E1 gene. Pair gene. In this way, a stable CAR receptor expression can be ensured without the use of a selection marker.
  • IRS interribosomal entry site
  • FIG. 6 Schematic representation of the p53-Ad5-E1 (final) plasmid.

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Abstract

L'invention concerne une lignée de cellules chorioniques immortalisées et un procédé pour produire des lignées de cellules immortalisées à partir de tissu chorionique embryonnaire humain ainsi que l'utilisation de la lignée cellulaire pour la production de protéine recombinante et/ou de substances actives médicales.
PCT/EP2014/063866 2013-07-01 2014-06-30 Lignée de cellules chorioniques humaines immortalisées ainsi que procédé pour immortaliser des cellules chorioniques humaines Ceased WO2015000856A1 (fr)

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