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WO1992007080A1 - Vecteur d'expression et de clonage d'adn episomique eucaryotique - Google Patents

Vecteur d'expression et de clonage d'adn episomique eucaryotique Download PDF

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Publication number
WO1992007080A1
WO1992007080A1 PCT/US1991/007690 US9107690W WO9207080A1 WO 1992007080 A1 WO1992007080 A1 WO 1992007080A1 US 9107690 W US9107690 W US 9107690W WO 9207080 A1 WO9207080 A1 WO 9207080A1
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vector
dna
cell
segment
transfected
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PCT/US1991/007690
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Roger R. Hewitt
Michael J. Siciliano
Randy J. Legerski
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Board Of Regents, The University Of Texas System
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Publication of WO1992007080A1 publication Critical patent/WO1992007080A1/fr

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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
    • 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
    • C12N15/63—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors

Definitions

  • This invention relates to the development of recombinant eu aryotic cloning and expression vectors based on unique regulatory elements isolated from autonomously replicating, stable episomal units isolated from human tumor cell lines. More specifically, the unique regulatory elements include origins of DNA replication, and DNA sequences that confer extrachromosomal stability and maintenance. These unique episomal regulatory elements permit large pieces of DNA to be expressed or cloned (greater than 50 kilobase pairs [kb] in size) .
  • the gene that is defective in Duchenne's muscular dystrophy is estimated to include more than a million base pairs (1000 kb) .
  • a striking feature of this gene is the protein-coding portion may be encoded by as little as 15 kb of DNA (Monaco, et al. Nature (London) . 302:575, 1983).
  • TNF tumor necrosis factor
  • Questions and concerns about this technology pertain to (1) the stability of the recombinants, (2) whether clone banks are representative of the starting material, (3) whether the desired protein is consistently expressed in extrachromosomal vectors, and (4) whether normal human transcripts are properly processed in yeast, as well as, whether proper expression and post translational modification of the recombinant protein occurs in yeast.
  • a cell can accumulate large amounts of specific protein or RNA is by amplification of the respective gene.
  • This amplification may be located on either expanded chromosomal regions (homogenous staining regions) or on extrachromosomal autonomously replicating elements (called double minute, double minute chromosomes or episomes) .
  • Episomes have unique features; the most notable are that episomes autonomously replicate and are stably maintained extrachromosomally. The characteristics of episomes permits the continuous production of the respective amplified gene and the gene products it encodes.
  • an episome produced in hamster cells has been characterized to contain amplified amounts of a transfected CAD (CAD is an acronym for the multifunctional protein containing carbamylphosphate synthetase, aspartate transcarbamylase, and dihydroorotase) gene at high frequency (Carrol, et al., Molecular and Cellular Biology. 7(5):1740, 1987).
  • the amplified CAD gene is produced with each division of each cell. Viral episomes have also been identified.
  • Circular bovine papilloma viru (BPV) DNA can transform certain mouse cell lines to a malignant phenotype. In these transformed cell lines, th BPV DNA remains circular and extrachromosomal at about 30 - 100 copies per cell. This "plasmid" is being stably maintained in higher eukaryotes. Desired genes may be inserted into the BPV DNA and be maintained in the plasmid-like state and high levels of mRNA and protein corresponding to the desired gene can be produced.
  • Epstein-Barr virus vectors contain sequences that provide extrachromosomal stability of episomal DNA as well as origins of replication.
  • This viral vector has been used to identify human DNA sequences that permit autonomous replication in human cells (Krysan, et al., Molecular and Cellular Bioloct ⁇ . 9(3):1026, 1989).
  • there are many limitations when working with a virally produced protein For example, in terms of producing proteins that may ultimately be used to replace defective human genes, viral episomes probably are not feasible because of potential Food and Drug Administration regulations, etc. Also the viral episome eventually integrates into chromosomal sites which then interferes with continued amplification and causes the expression of its resident genes to be extinguished.
  • This minimal cloning or expression vector will be further modified by the inclusion of regions of human chromosomes containing telomeres and centromeres. This would thus create a human artificial chromosome that would be subjected to the same control mechanisms (regarding regulation and chromosomal segregation) as normal chromosomes and therefore serve as a vehicle for gene replacement therapy.
  • This modification of the extrachromosomal vector is therefore unique in that it will be a synthetic chromosome containing genes of choice, that will be expressed, and that will be maintained and regulated as if it were a normal chromosome.
  • This cloning or expression vector may take on several forms.
  • two principal forms for employment are: (1) employed via extrachromosomal/episomal, autonomous replication and segregation which could even be amplified, and (2) employed via a human artificial chromosome under normal chromosomal control mechanisms.
  • the present invention relates to the development of recombinant eukaryotic cloning and expression vectors based on unique regulatory elements isolated from autonomously replicating, stable episomal units isolated from human tumor cell lines. More particularly, these unique regulatory elements include origins of DNA replication, and DNA sequences that confer extrachromosomal stability and maintenance. These unique episomal regulatory elements will permit large pieces of DNA to be expressed or cloned (greater than 50 kilobases pairs in size) .
  • cloning vector refers to a DNA vector designed to be used to clone a desired gene.
  • the techniques that are involved in cloning vary from vector to vector and from system to system, however, these techniques in general are standard and known to those skilled in the art of recombinant DNA technology.
  • expression vector refers to a DNA vector capable of replication in selected mammalian host cells and expressing a desired protein. This protein may then be recovered from the cells by employing techniques known to those skilled in the art.
  • This cloning vector should include one or more functional origins of DNA replication to permit stable, autonomous replication.
  • the phrase "origin of replication" is defined as a region that indicates the origin of replication.
  • This cloning vector should include appropriate DNA sequences that confer extrachromosomal stability and maintenance.
  • the sequences responsible for conferring extrachrom -o al stability and persistence may be related to sequences responsible for nuclear matrix attachment sites, topoisomerase II reaction sites, and/or other regions required for appropriate interactions with the nuclear architecture. This extrachromosomal stability and maintenance permits the introduction of large exogenous genes.
  • This cloning vector should also include DNA selectable marker sequences that can be used to confer drug resistance to a transfected cell or DNA sequences that can correct a genetic mutation. This allows the cells that were transfected with the vector to be selected for. The DNA selectable marker segment confers upon a cell transfected with said vector, the ability to survive in the presence of a selected compound or selected group of compounds.
  • the compound may be either G418 or hygromycin B.
  • selectable marker segments will contain DNA encoding an enzyme capable of functionally replacing a mutated enzyme so as to render the transfected cell resistant to said selected compound or selected group of compounds.
  • the enzyme may be selected from a group consisting of: thymidine kinase, xanthineguanine phosphoribosyl transferase, adenine phosphoribosyltransferase, adenosine deaminase and dihydrofolate reductase.
  • This cloning vector should also include a multi-use multiple cloning site to facilitate recovery for genetic modification and analysis and insertion for reintroduction into cells for replication and expression.
  • Multiple cloning cassette sequence cartridges are commercially available from several different companies (Promega, New England Biolabs, etc) .
  • a typical cassette sequence would include restriction sites for 8 - 11 different enzymes (i.e. Eco RI, Sac 1, Sma 1, Ava I, Bam HI, Xba 1, Hinc II, Ace 1, Sal 1, Pst 1, Hind III, etc.) The availability of these cassette sequences are known to those skilled in the art.
  • This cloning vector should also include a DNA segment encoding bacterial components necessary for propagation of said vector in bacteria.
  • Bacterial components that are essential for propagation of the cloning vector in bacteria are known to those skilled in this art.
  • two bacterial components essential for bacterial propagation are a replicon that is responsible for initiation of replication and antibiotic resistant markers (i.e. ampicillin, tetracycline, etc.) that permits growth in specific antibiotics.
  • a unique expression vector capable of expressing large pieces of DNA should also include, a promoter, a polyadenylation site and a splice site in spacial relation to allow efficient expression of a structural gene.
  • promoters to be included in this vector will depend on the mammalian host cell employed. It is advantageous to employ a compatible promoter with regard to the cells that the desired protein will be expressed in.
  • the inventors prefer to employ promoters derived from the following genes (although other promoters would be satisfactory) : cytomegalovirus, SV-40, Rous sarcoma virus, thymidine kinase, beta-actin, metallothionein, and the epidermal growth factor receptor gene isolated from a DiFi episome.
  • a polyadenylation site refers to the site at which a poly A tail (a stretch of 50 to 300 adenines) is added to the vector for efficient expression of a desired protein in a mammalian cell.
  • splice site refers to a bacterial processing site essential to remove introns incorporated into the bacterial plasmid. These components are essential for optimal expression of a desired protein.
  • a further embodiment of this invention is an artificial chromosome consisting of a DNA segment derived from a non-viral episome, said segment containing an origin for DNA replication, a DNA segment derived from a non-viral episome, said segment containing a DNA sequence which confers upon said vector the ability to be stably maintained extrachromosomally in a cell transfected with said vector, a DNA segment containing a multiple cloning site, a DNA selectable marker segment conferring upon a cell transfected with said vector, the ability to survive in the presence of a selected compound or selected group of compounds, a DNA segment encoding bacterial components necessary for propagation of said vector in bacteria, a promoter, a polyadenylation site, a splice site, a DNA segment encoding a centromere and a DNA segment encoding a telomere.
  • a substantially purified non-viral episome of human origin capable of stable extrachromosomal maintenance and of autonomous replication in a compatible mammalian cell line.
  • a substantially purified episomal DNA segment containing an origin of replication This invention further includes a substantially purified* episomal DNA segment containing a DNA sequence, which confers upon a vector including said segment, the ability to be stably maintained extrachromosomally in a cell transfected with said vector.
  • a DNA segment containing an origin for DNA replication is from an episome isolated from DiFi colorectal cell line.
  • Another embodiment of this invention is a DNA sequence which confers upon said vector the ability to be stably maintained extrachromosomally in a cell transfected with said vector is from an episome isolated from DiFi colorectal cell line.
  • Another embodiment of this invention is a DNA segment containing an origin for DNA replication and a DNA sequence which confers upon said vector the ability to be stably maintained extrachromosomally in a cell transfected with said vector is from an episome isolated from DiFi colorectal cell line.
  • operatively spaced with respect to a desired gene is defined as the appropriate positional spacing required between the numerous cloning and expression vectors components described in this invention so as to allow each of the of components to achieve its desired function.
  • transfecting eukaryotic cells with these unique cloning or expression vectors the transfection techniques are standard and known to those skilled in the art of recombinant DNA technology.
  • this invention could also be applied for the production of stable cell lines which are, by definition, continuously producing the desired protein. The production of cell lines designed to continuously produce the desired protein has been described extensively in the literature, and is therefore known to those skilled in the art.
  • CHARACTERISTICS OF THE DEPOSITED CELL LINE Cell line "DiFi" comprising cells obtained from the ascitic fluid of a colorectal tumor in a patient with
  • Gardner's syndrome is available from the ATCC, accession ⁇ CRL 10576. This cell line retains 50 copies or more of extrachromosomal episomes, each of which contains at least one complete copy of the epidermal growth factor receptor gene.
  • Fig. 1 In situ hybridization of DiFi cells with EGFR.
  • A A portion of a metaphase from DiFi cells stained with Giemsa
  • B fluorescence visualization of in situ hybridization using biotinylated EGFR as probe and counterstained with propidium iodide
  • C black and white print of the fluorescence pattern of in situ hybridization
  • Fig. 2 Electrophoretic mobilization of EGFR ⁇ enes by ⁇ aroma irradiation. Autoradiogram of a Southern blot of a TAFE gel hybridized with 32P-labeled EGFR. Origin (o) is indicated at the top as is the direction of migration. Plug samples 1-8 were exposed to 0, 5, 10, 20, 40 80, 160, 320 Gray, respectively. Hybridization membranes were exposed to film for 24 hrs.
  • FIG. 3 Effect of ⁇ aroma irradiation on the electrophoretic mobilization EGFR in A431.
  • DiFi. and HeLa cells Autoradiogram of a Southern blot of a TAFE gel hybridized with 32P-labeled EGFR. Origin and direction of migration is as in Fig. 2.
  • A431, DiFi and HeLa cell DNA plugs were irradiated with A. OGy, B. 10 Gy, C. 40 Gy, D. 160 Gy. Autoradiographic exposure was extended to 72 hr in order to enhance sensitivity for detecting any fragments that might have migrated from the A431 plugs.
  • Fig. 4 CHEF analysis of EGFR in gamma irradiated DiFi. Plugs containing DiFi DNA were exposed to 31.4 Gy prior to electrophoresis. The analysis of control (c) and irradiated (R) samples was performed in duplicate. Approximate sizes of the observed fragments, in kbs, are indicated to the right.
  • the episomes are isolated from the origin in a substantially purified form and the minimal essential elements for episomal replication and transcription are localized and isolated. Those elements are then ligated into a selected DNA molecule, together with additional DNA segments, including, for example, selectable markers, multiple cloning site or sites, segments necessary for propagation in bacteria and/or a promoter enhancer, splice site and polyadenylation site.
  • Replication of nuclear DNA in eukaryotes appears to be under precise and reproducible control, such that it is replicated only once in each S-phase, the DNA synthetic portion of each cell division cycle.
  • each portion of the genome replicates at the same time in each S-phase, with expressing (transcribed) genes replicating early and non-expressing and/or structural DNA replicating late.
  • prokaryotes, viruses, and yeast contain DNA sequences called origins, that serve as sites for initiating cycles of DNA replication.
  • origins DNA sequences that serve as sites for initiating cycles of DNA replication.
  • sites define replicating units, or replicons, in eukaryotic cells such as human cells.
  • This invention exploits these cell lines by isolating and investigating the structure and replication control of their extrachromosomal elements in order to identify DNA sequences required to ensure their autonomy for stable maintenance, replication and gene expression.
  • This minimal essential structure should then provide the core structure with which to assemble a cloning and expression vector for genes exceeding sizes accommodated by cosmid vectors.
  • DiFi cells were (1) successfully established in tissue culture, (2) shown to contain amplified EGFR genes and mRNA, and (3) characterized cytologically to be near tetraploid with the presence of double minutes (dmin; Bowman et al. In Hereditary Colorectal Cancer. J. Utsunomiya and H. Lynch (eds) , SpringVerlag, In Press, 1990) .
  • Malignant ascitic fluid cells were isolated from a 46 year old female rectal cancer patient with Gardner syndrome and initiated to grow in culture. The cells have been maintained in culture for over three years. Hoechst stain analysis for mycoplasma was negative. Subcutaneous injection of DiFi cells into athymic mice demonstrated tumor production in 50% of the mice. The cells have a tetraploid karyotype, and possess an isozyme pattern characteristic of colorectal cancer cell lines.
  • Slides containing metaphase cells from either DiFi or SW480 cells were prepared and stored at room temperature. Prior to in situ hybridization with a biotinylated EGFR probe, the slides were stained (six minutes in 5% Giemsa prepared in phosphate buffer pH 6.8) and photographed. In situ hybridization involved treating the photographed slides with RNAse, DNA denaturation and dehydration solutions, overnight incubation in a hybridization mix containing a biotinylated EGFR probe, and tagging the regions of EGFR hybridization with fluorescein-avidin and biotinylated goat anti-avidin.
  • Giemsa-stained metaphase chromosomes from DiFi cells revealed a background of extrachromosomal particles at the limit of optical resolution (Fig. 1A) . Occasionally, they were paired in the form of small dmins. To determine whether these structures contained copies of the EGFR gene, the biotinylated A64-3 cDNA EGFR probe was hybridized to these metaphase cells.
  • SW480 cells served as a negative control because their dmins are amplified for MYC rather than EGFR (Untawale, Masters Thesis on File at the graduate School'of Biomedical Sciences, University of Texas Health Science Center, Houston, Texas, 1987; Untawale and Singh, Anticancer Res. 8:1-8, 1988).
  • IB Fluorescence visualization of in situ hybridization using biotinylated EGFR as a probe and counterstained with propidium iodide is shown in IB, and a black and white print of the fluorescence pattern of in situ hybridizatio is shown in 1C.
  • the chromosomes are intensely stained in contrast to the diffuse staining of extrachromosomal material in the background.
  • the extrachromosomal background appears to be dmin, which var in their size and visibility.
  • Hybridization of the biotinylated EGFR probe was limited to extrachromosomal regions containing dmin, rather than chromosomal DNA (IB) .
  • IB chromosomal DNA
  • IB chromosomal DNA
  • Figure 1C the extrachromosomal labeling was visualized more clearly since the fluorescein fluorescence is more intense in dmi than isothe propidium fluorescence from the chromosomes.
  • the inventors exposed agarose plugs containing unsheared DiFi cellular DNA to varying doses of gamma radiation prior to analysis by pulse-field gel electrophoresis. Appropriate levels of exposure were estimated based on an expected yield of 1.1 x 10" 8 double-strand breaks/Gy/bp (calculated from Krisch et al., Rad. Res. 101:356-372, 1985).
  • FIG 2 an autoradiogram of a Southern blot of a TAFE gel probed with 32P-labeled EGFR. demonstrates electrophoretic mobilization of EGFR genes by gamma irradiation. The origin (o) as well as the direction of migration is indicated at the top of the figure. Plug samples 1-8 were exposed to 0, 5, 10, 20, 40 80, 160, 320 Gy, respectively. Hybridization membranes were exposed to film for 24 hrs.
  • FIG. 3 an autoradiogram of a Southern blot of a TAFE gel probed with 32P-labeled EGFR. demonstrates the effect gamma irradiation has on the electrophoretic patterns of migration of EGFR sequences in A431, DiFi, and HeLa cells. The origin and direction of migration are as in Fig. 2. DNA plugs from A431, DiFi and HeLa cells were irradiated with increasing amounts of radiation: Lane
  • EGFR amplification is much higher in DiFi DNA and A431 DNA when compared to HeLa DNA. More importantly, sample plug irradiation did not release discrete sizes of HeLa and A431 EGFR sequences were (confirmed by exposing autoradiograms for 7 days, data not shown) . However, mobilizatior of both the 650 kb band and 1300 kb band DiFi EGFR fragments were readily detected. To summarize, EGFR sequences in both HeLa and A431 DNA appear to be chromosomally localized. In contrast, EGFR sequences in DiFi DNA appear to be episomally (extrachromosomally) localized and may be substantially purified by the procedure described here.
  • Figure 4 presents CHEF analysis of EGFR from gamma irradiated DiFi DNA. Plugs containing DiFi DNA were exposed to 31.4 Gy prior to electrophoresis. The analysis of control (c) and irradiated (R) samples was performed in duplicate. Approximate sizes of the observed fragments, in kbs, are indicated to the right. Irradiating DiFi plugs and conducting CHEF electrophoresis under conditions that resolve larger DNA fragments revealed the presence of a weakly hybridizing band of approximately 2,000 kb, in addition to the 650 kb and 1300 kb fragments (Fig. 4) .
  • the identification, characterization and isolation of DNA regulatory regions within the episomes that function a) as origins of autonomous DNA replication, and b) function as stabilizing regions for extrachromosomal maintenance will permit the construction of cloning and expression vectors that replicate and function as extrachromosomal vectors.
  • the following is meant to serve as one example of identifying and isolating such regulatory factors from the episomal unit maintained in human tumor cell. In some instances, reference is made t working with the episomal unit from DiFi cells; DiFi is used here only as an example.
  • the ideal starting point is a preparation that is highly enriched for the episomes of interest.
  • a highly enriched source of EGFR-containing episomes is the human DiFi cell line. DNA will be isolated from this enriched preparation and most of the DiFi genomic DNA can be eliminated from this preparation by employing an alkaline lysis modification (Griffin, et al., J. Virol.. 40:11-19, 1981) .
  • An essentially pure preparation of DiFi episomes can then be obtained by preparative electrophoresis on agarose gels that permits the mobilization of covalent circular DNA molecules (Carroll et al., Mol. Cell. Biol.. 7:1740-1740 (1987)). These "olecules can then be recovered from the gels by procedures that dissolve or digest (agarose) the agarose and permit the episomal DNA to be purified directly from the digest.
  • a restriction enzyme analysis will be performed after the episome is isolated. For example, most of the DiFi episome can be separated into two pieces by exploiting the limited number of sites susceptible to restriction enzymes Mlul (2 sites) and NotI (2 sites) . Mlul cuts at two closely spaced sites whereas NotI cuts at two widely distant sites. Table 1 presents macrorestriction fragment sizes of DiFi episomes digested with Mlul and NotI restriction enzyme.
  • Lambda libraries were constructed that represented 2 to 10 kb portions of the DiFi episome by utilizing partially restriction enzyme digested episomes or NotI fragments and the Lambda-Zap phagemid vector (Short, Fernandez, Sorge, and Huse, Nuc. Acids Res. 16:7583-7600, 1988) .
  • Cosmid libraries are constructed with Ba HI partial digests of isolated episomes or NotI DiFi episomal fragments by utilizing the sCosl vector (Evans, et al. Gene. 79:9-20, 1989). These cosmid libraries represent portions of the DiFi episome in approximately 40 kb blocks. 3. PI Libraries
  • Recombinant DNA libraries containing portions of the DiFi episome are constructed by utilizing the PI bacteriophage based cloning vector (Sternberg, Proc. Nat. Acad. Sci. USA. 87:103-107, 1990).
  • This PI library contains DiFi episomal portions representing two size ranges: less than 30 kb and approximately 85 - 110 kb.
  • DiFi episome are constructed utilizing an E. coli F sex factor based cloning vector (Leonardo and Sedivy, Biotechnology. 8:841, 1990).
  • This F plasmid library contains DiFi episomal portions up to at least 150 kb.
  • other plasmid libraries can be constructed using one of several available plasmid vectors (i.e. pKS, pT7 ⁇ T3a-18, etc.). These vectors are known to those skilled in this art.
  • plasmid vector clones from the DiFi Cosl library are introduced into for example, HSF56 human primary fibroblast cells via calcium phosphate transfection or electroporation.
  • Each Cosl vector clone contains a selectable marker that confers drug resistance to G418, for example.
  • Retention and replication of transfected clones are identified by growing the transfected population of HSF56 cells in the presence of G418, a compound which specifically selects for cells that are neomycin resistant. The cells are placed under G418 selection 2 days after transfection, and G418 resistant populations are grown for at least two months by maintaining the resistant clones appropriate subculturing techniques known to those skilled in the art of tissue culture.
  • Neomycin resistant clones that persist for several cell divisions therefore contain a DiFi Cosl vector clone that is replicating.
  • a persistent neomycin resistant cell clone is recovered and low molecular weight DNA (less than 120 kb) is isolated by the HIRT extraction method (Hirt, J. Mol. Biol.. 26:265-369, 1967).
  • the DNA isolated from this neomycin resistant cell clone will be subcloned into plasmid vectors that accommodate smaller inserts, such as the pKS vector or the pT7/T3a-18 vector, which, preferably, will also contain a selectable marker, such as a gene encoding beta lactamase, which confers resistance to ampicillin.
  • the result of this will be another plasmid library which includes specific regions, one or more of which contain an origin for DNA replication.
  • the clones from this new library will next be introduced into bacteria and bacterial colonies resistant to, for example, ampicillin, will be isolated.
  • the host is an E. coli cell of a type which is compatible with the vector type.
  • the DNA from the ampicillin resistant bacterial colonies will be transfected into a mammalian cell line.
  • the DNA (isolated with the HIRT extraction method) from the transfected mammalian cells will be analyzed by the Dpn I digestion (Krysan et al., Molec. Cell. Biol. 9:1026-1033, 1989 which is incorporated herein by reference) .
  • DNA exhibiting the bacterial methylation pattern is cleavable by Dpn I restriction enzyme while DNA with mammalian methylation pattern is not.
  • DNA that is not digested by Dpn I has replicated in the mammalian cell.
  • the origins for DNA replication will then be identified within the inserts in autonomously replicating clones.
  • the origin can then be removed from the vector, and inserted into the recombinant cloning vector.
  • Vectors that include regions from the DiFi episome are designated pDFE ori + and will serve as the recipients for inclusion of other regions of the DiFi episome conferring episome maintenance.
  • Identifying those individual clones that contain a region conferring extrachromosomal stability is determined by long term culturing (longer than two months) in the presence of a selection drug.
  • the clones that survive the continuous exposure to the selection drug must contain a region that confers extrachromosomal stability.
  • clones that persist during several cell division cycles will also be evaluated to identify regions within episomal DNA that confer stability for maintenance of extrachromosomal molecules.
  • the procedure by which isolation of this region is essentially the same one as described for identifying the replication region, except that vectors containing DiFi episomal origins of replication will be used to clone other restriction fragments from the DiFi episome.
  • the episomal DNA may be isolated and introduced into bacteria and bacterial colonies resistant to, for example, ampicillin, will be isolated.
  • the DNA from the ampicillin resistant bacterial colonies will be transfected into a mammalian cell line.
  • the DNA (isolated with the HIRT extraction method) from the transfected mammalian cells will be analyzed for fragment size and, depending on that size, another cycle may be initiated to further reduce the size of the piece of DNA that confers the extrachromosomal stability.
  • vector-containing cells are treated with the non-ionic detergent Triton X-100 and 2M NaCl. This treatment produces salt extracted residual nuclei, called nucleoids, which can be centrifuged into a pellet at low speeds.
  • nucleoids salt extracted residual nuclei
  • Vectors associated with the nuclear matrix will pellet with the nucleoids; if they do not pellet with the nucleoids they will remain in the extracts' supernate.
  • c. a DNA or genomic DNA region encoding bacterial components necessary for propagation of the vector in bacteria.
  • Selectable markers for mammalian cells, confer resistance to a specific selection agent once DNA conferring the resistance is transfected into individual cells possessing a genetic inheritance pattern appropriate for the selectable marker being used in the vector.
  • selectable marker for mammalian cells, confer resistance to a specific selection agent once DNA conferring the resistance is transfected into individual cells possessing a genetic inheritance pattern appropriate for the selectable marker being used in the vector.
  • selection agents There are a variety of different dominant and recessive selection agents known to those skilled in the art. Any one of the following genes and agents should be effective in terms of employing a selection system:
  • ⁇ G418 resistance is selected by exposure to medium containing 100 to 800 ug/ml G418.
  • G418 selects for cells deficient in the enzyme aminoglycoside phosphotransferase and are referred to as neomycin resistant cells.
  • HAT resistance for forward selection (converting a thymidine kinase minus cell to a thymidine kinase positive cell) is selected with complete medium supplemented with 100 uM hypoxanthine, 0.4 uM aminopterin, 16 uM thymidine and 3 uM glycine.
  • HAT medium selects for variants defective in either hypoxanthine-guanine phosphoribosyl-transferase or thymidine kinase (Littlefield, Proc. Natl. Acad. Sci. USA. 50:568, 1963; Littlefield, Science. 145:709-710, 1964).
  • Hygromycin B resistance is selected by exposure to complete medium supplemented with 10 - 400 ug/ml hygromycin B.
  • Hygromycin B selects for variants defective in the enzyme hygromycin-B-phosphotransferase (Gritz and
  • D Adenine phosphoribosyltransferase (APRT) positive variants are selected by exposure to medium supplemented with 25 uM alanosine, 50 uM azaserine and 100 uM adenine (Lowy, et. al. , Cell. 22:817, 1980; Adair, et. al., Proc. Natl. Acad. Sci. USA. 86:4574-4578, 1989).
  • Phosphoribosyltransferase (XGPRT) positive variants are selected with complete medium supplemented with dialyzed fetal calf serum, 250 ug/ml xanthine, 15 ug/ml hypoxanthine, 10 ug/ml thymidine, 2 ug/ml aminopterin, 25 ug/ml mycophenolic acid, and 150 ug/ml L-glutamine (Mulligan and Berg, Proc. Natl. Acad. Sci. USA. 78:2072-2076, 1981).
  • Methotrexate resistance is selected by exposure to complete medium supplemented with 0.01 uM - 300 uM methotrexate and dialyzed fetal calf serum. Methotrexate selects for cells expressing high levels of dihydrofolate reductase (O'Hare, et al., Proc. Natl. Acad. Sci. USA. 78:1527, 1981; Simonsen and Levinson, Pro. Natl. Acad. Sci. USA. 80:2495-2499, 1983).
  • D Deoxycoformycin resistant cells are selected by exposure to complete medium supplemented with 10 ug/ml thymidine, 15 ug/ml hypoxanthine, 4 uM 9-B-D- xylofuranosyl adenine (XylA) , and 0.01 - 0.03 uM 2'-deoxycoformycin (dCF) .
  • This selection selects for mutants expressing adenosine deaminase (ADA; Kaufman, et. al., Proc. Natl. Acad. Sci. USA, 83:3136-3140, 1986).
  • this optimum eukaryotic cloning vector could include a DNA region comprising a multiple cloning cassette sequence containing infrequent cutting by restriction enzymes to facilitate the insertion of a desired gene.
  • Multiple cloning cassette sequence cartridges are commercially available from several different companies (Stratagene, Promega, New England Biolabs etc) .
  • a typical cassette sequence cartridge would inc _ ⁇ de restriction sites for 8 - 11 different enzymes (i.e. Eco Rl, Sacl, Sma 1, Ava 1, Bam HI, Xba 1, Hinc II, Ace 1, Sal 1, Pst 1, Hind III, etc.).
  • Eco Rl, Sacl, Sma 1, Ava 1, Bam HI, Xba 1, Hinc II, Ace 1, Sal 1, Pst 1, Hind III, etc. The availability of these cassette cartridges are known to those skilled in the art.
  • the bacterial plasmid sequences may be derived from any one of the many different vectors that are commercially available and known to those skilled in the art of recombinant DNA technology.
  • pUC, pKS, pBR322 and pT7/T3al8 are used as a matter of preference, however, other vectors would be equally effective.
  • pBR322 sequences are introduced into the cloning or expression vector, the resulting recombinant can then be shuttled back and forth between E. coli and mammalian cells.
  • an optimal eukaryotic expression vector that can accommodate 40 kb - 400 kb pieces of DNA will also contain, in addition to the elements described for the cloning vector, a DNA region containing a promoter, a polyadenylation and splice site necessary for the expression of the desired gene.
  • the first and more simpler approach is to begin with a readily available cloning plasmid vector capable of propagation in bacteria.
  • cloning plasmid vector capable of propagation in bacteria.
  • vectors known to those skilled in the art that would work efficienty.
  • Several different components and features can easily be ligated into this bacterial plasmid vector. These added features are discussed below.
  • the vector will not only have the core structure (to confer the ability to replicate DNA and to be maintained extrachromosomally) but will also have the added features to optimize the vector for propagation in bacteria and for identification of its presence after transfection into a mammalian cell recipient.
  • the second approach involves custom designing andcreating the optimum cloning vector by ligating all the desired features and components (including the core structure) together to generate the vector of choice.
  • the episomally maintained and replicated vector pDFE ori + mat + is introduced into cells and persist as covalent circular extrachromosomal molecules. In this form the episomes accumulate to produce multiple copies in each cell and accordingly, also overproduce mRNA and its protein product. While this is desirable for producing amplified genes and gene products, the introduction of cloned genes into cells for use in gene therapy requires the control of gene copy number and attendant gene expression. Such control is introduced into the DiFi episome vector by introducing DNA sequences that stabilize artificial chromosomes containing linear double stranded DNA (DNA encoding a telomere) .
  • telomere sequences occur at the termini of natural chromosomes; in human chromosomes 5'-AGGGTT-3' is tandemly repeated to the extent of 10 of 15 kb at every telomere (Blackburn, Science. 249:489, 1990) .
  • This tandemly repeated sequence is ligated to each end of a linearized cloning and expression vector to stabilize the termini.
  • telomere sequences specific for other species provides for the stabilization of artificial chromosomes when introduced into those species.
  • Centromere sequences are known to identify regions within chromosomes where kinetichores are organized and mitotic spindles are attached to the chromosomes, thus ensuring for the segregation of chromosomes during mitosis.
  • DNA sequences that serve as centromeres are introduced into an internal region of the linearized cloning and expression vector which contain telomeres resulting in an artificial chromosome.
  • This synthetic chromosome contains required regulatory and stabilizing DNA sequences that normally occur in natural chromosomes.
  • this synthetic chromosome by ligating a gene of interest into its multiple cloning site.
  • the gene or cDNA derivative of the gene that is defective in Duchenne's muscular dystrophy or myotonic dystrophy, or one of a number of other diseases associated with muscle dysfunction may be cloned into the artificial chromosome.
  • the artificial chromosome is then introduced into cells or tissues or animals by methods appropriate for the target.
  • the transfected chromosome is established as an integral component of the recipient cells where it is stably maintained and expressed. Recipient cells, tissues or animals that were initially dysfunctional because of a genetic defect they possessed are cured and become normal because of the expression and synthesis of the normal gene product introduced in the artificial chromosome.
  • Mammalian Cells Once the optimal cloning and expression vector is constructed, several different strategies for transfecting the vectors will be studied. Examples of potential methods includes: (1) encapsulation of insert-containing vectors in liposomes of appropriate composition to enhance entry into target cells, and (2) electroporation of vector into mitotic cell recipients to enhance its inclusion within the nucleus as cells progress into Gl phase of the cell cycle, and (3) injection of DNA-encoated particles into cells by employing a Biolistic Particle Delivery System (DuPont) . This procedure essentially shoots DNA-coated bullets into cells or tissues.
  • DuPont Biolistic Particle Delivery System

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Abstract

L'invention se rapporte au développement de vecteurs d'expression et de clonage eucaryotiques recombinants basés sur des éléments régulateurs uniques isolés à partir d'unités épisomiques stables à réplication autonome de lignées cellulaires tumorales humaines. Plus particulièrement, les éléments régulateurs uniques se rapportent à des régions d'origine de réplication, tout en favorisant la stabilité et la maintenance extrachromosomiques. Ce vecteur d'expression et de clonage peut contenir des gènes qui dépassent la limite cosmide (supérieure à 50 kb) et permet leur maintenance sous forme d'éléments extrachromosomiques à réplication autonome dans des cellules mammifères. L'inclusion de télomères et de centromères pourrait réguler la réplication et la ségrégation et servir ainsi de véhicule éventuel pour la thérapie de remplacement des gènes. Cette invention est par conséquent unique en ce qu'elle permet l'expression et la réplication autonome de larges fragments de gènes, maintenus de manière extrachromosomique, dans un vecteur contenant des éléments régulateurs épisomaux.
PCT/US1991/007690 1990-10-17 1991-10-16 Vecteur d'expression et de clonage d'adn episomique eucaryotique WO1992007080A1 (fr)

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WO1996040965A1 (fr) * 1995-06-07 1996-12-19 Case Western Reserve University Chromosome synthetique de mammifere et procedes de construction de celui-ci
US5698686A (en) * 1994-10-20 1997-12-16 Arch Development Corporation Yeast telomerase compositions
WO1998008964A1 (fr) * 1996-08-26 1998-03-05 Tsuneko Okazaki Chromosomes artificiels de mammifere
WO1998027200A3 (fr) * 1996-12-16 1998-10-01 Univ Mcgill Sequences consensus de l'origine de replication d'adn humain et d'adn de mammifere
US5869294A (en) * 1995-06-07 1999-02-09 Case Western Reserve University Method for stably cloning large repeating DNA sequences
WO1998053056A3 (fr) * 1997-05-17 1999-02-25 Dirk Schindelhauer Procede de preparation de produits de synthese d'adn longs
US6743967B2 (en) 1996-04-10 2004-06-01 Chromos Molecular Systems Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US7119250B2 (en) 1997-06-03 2006-10-10 The University Of Chicago Plant centromere compositions
US7193128B2 (en) 1997-06-03 2007-03-20 Chromatin, Inc. Methods for generating or increasing revenues from crops
US7227057B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
US7235716B2 (en) 1997-06-03 2007-06-26 Chromatin, Inc. Plant centromere compositions
US7847151B2 (en) 1997-06-03 2010-12-07 The University Of Chicago Plant artificial chromosome (PLAC) compositions and methods
US7989202B1 (en) 1999-03-18 2011-08-02 The University Of Chicago Plant centromere compositions
US8222028B2 (en) 2005-09-08 2012-07-17 Chromatin, Inc. Plants modified with mini-chromosomes
US8350120B2 (en) 2004-02-23 2013-01-08 The Univesity of Chicago Plants modified with mini-chromosomes
US8614089B2 (en) 2007-03-15 2013-12-24 Chromatin, Inc. Centromere sequences and minichromosomes
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes

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US5916752A (en) * 1994-10-20 1999-06-29 Arch Development Corporation Telomerase screening methods
US5698686A (en) * 1994-10-20 1997-12-16 Arch Development Corporation Yeast telomerase compositions
US6387619B1 (en) 1994-10-20 2002-05-14 Arch Development Telomerase compositions and methods
US6348353B1 (en) 1995-06-07 2002-02-19 Case Western Reserve University Artificial mammalian chromosome
US5869294A (en) * 1995-06-07 1999-02-09 Case Western Reserve University Method for stably cloning large repeating DNA sequences
AU724695B2 (en) * 1995-06-07 2000-09-28 Case Western Reserve University Synthetic mammalian chromosome and methods for construction
WO1996040965A1 (fr) * 1995-06-07 1996-12-19 Case Western Reserve University Chromosome synthetique de mammifere et procedes de construction de celui-ci
US6743967B2 (en) 1996-04-10 2004-06-01 Chromos Molecular Systems Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US6297029B1 (en) 1996-08-26 2001-10-02 Tsuneko Okazaki Mammalian artificial chromosomes
EP2060633A1 (fr) * 1996-08-26 2009-05-20 Chromo Research, Inc. Chromosomes artificiels de mammifères
WO1998008964A1 (fr) * 1996-08-26 1998-03-05 Tsuneko Okazaki Chromosomes artificiels de mammifere
US6569643B2 (en) 1996-08-26 2003-05-27 Tsuneko Okazaki Mammalian artificial chromosomes
WO1998027200A3 (fr) * 1996-12-16 1998-10-01 Univ Mcgill Sequences consensus de l'origine de replication d'adn humain et d'adn de mammifere
US6410722B1 (en) 1996-12-16 2002-06-25 Mcgill University Human and mammalian data replication origin consensus sequences
WO1998053056A3 (fr) * 1997-05-17 1999-02-25 Dirk Schindelhauer Procede de preparation de produits de synthese d'adn longs
US7119250B2 (en) 1997-06-03 2006-10-10 The University Of Chicago Plant centromere compositions
US8759086B2 (en) 1997-06-03 2014-06-24 University Of Chicago Methods for generating or increasing revenues from crops
US7226782B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
US7235716B2 (en) 1997-06-03 2007-06-26 Chromatin, Inc. Plant centromere compositions
US7456013B2 (en) 1997-06-03 2008-11-25 Chromatin, Inc. Plant centromere compositions
US7193128B2 (en) 1997-06-03 2007-03-20 Chromatin, Inc. Methods for generating or increasing revenues from crops
US7847151B2 (en) 1997-06-03 2010-12-07 The University Of Chicago Plant artificial chromosome (PLAC) compositions and methods
US7227057B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
US8062885B2 (en) 1997-06-03 2011-11-22 The University Of Chicago Plant centromere compositions
US7989202B1 (en) 1999-03-18 2011-08-02 The University Of Chicago Plant centromere compositions
US8350120B2 (en) 2004-02-23 2013-01-08 The Univesity of Chicago Plants modified with mini-chromosomes
US8729341B2 (en) 2004-02-23 2014-05-20 University Of Chicago Plants modified with mini-chromosomes
US8222028B2 (en) 2005-09-08 2012-07-17 Chromatin, Inc. Plants modified with mini-chromosomes
US8614089B2 (en) 2007-03-15 2013-12-24 Chromatin, Inc. Centromere sequences and minichromosomes
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes

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