EP1409700A2 - Systeme destine a produire des populations clonales ou complexes d'adenovirus recombines et leur application - Google Patents

Systeme destine a produire des populations clonales ou complexes d'adenovirus recombines et leur application

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Publication number
EP1409700A2
EP1409700A2 EP02764728A EP02764728A EP1409700A2 EP 1409700 A2 EP1409700 A2 EP 1409700A2 EP 02764728 A EP02764728 A EP 02764728A EP 02764728 A EP02764728 A EP 02764728A EP 1409700 A2 EP1409700 A2 EP 1409700A2
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Prior art keywords
donor
cells
site
virus
population
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EP02764728A
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German (de)
English (en)
Inventor
Moritz Hillgenberg
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Develogen AG
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Develogen AG
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Priority to EP02764728A priority Critical patent/EP1409700A2/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
    • C12N15/86—Viral vectors
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • 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
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the invention relates to a novel system for the production of recombinant adenoviruses (rAd); Areas of application are medicine, veterinary medicine, biotechnology, genetic engineering and functional genome analysis.
  • genes into cells are relevant for several reasons.
  • the expression of genes introduced in cell culture systems enables e.g. the functional characterization of the encoded proteins or their production.
  • therapeutically effective genes represents a new way of treating human diseases (gene therapy).
  • a variety of approaches are being investigated to achieve medically or veterinarily effective immunization (vaccination) in humans and in livestock by transferring immunostimulatory and / or pathogen-specific genes.
  • immunization vaccination
  • the vector system must also offer the possibility of constructing complex gene banks.
  • Efficient gene transfer into cells is possible in particular with recombinant viral vectors which are derived from retroviruses, adeno-associated viruses or adenoviruses (overview in: Verma, M. and I. and Somia, N. (1 997) Nature 389, 239- 242).
  • the so-called E 1 -delegated adenoviral vectors of the first generation have been intensively researched as gene transfer vectors over the past decade (review by: Bramson, JL et al. (1 995). Curr. Op. Biotech. 6, 590-595).
  • adenovirus serotype 5 she derived from human adenovirus serotype 5 and are deleted in the essential E 1 region, often also in the non-essential E3 region, whereby up to 8 KBp of foreign DNA can be inserted into the virus genome.
  • These vectors can be produced to high titers on cells complementing the E 1 deficiency. Due to their high stability, they are easy to clean and store.
  • Recombinant adenoviruses have a broad spectrum of efficiently infectable cell types in vitro and also allow efficient gene transfer to different tissues in vivo. Clonal rAd populations are already used in many ways for gene transfer in vitro and in vivo.
  • This recombination of the two S ⁇ i / tt / e plasmids can take place after contransfection in 293 cells (McGrory, WJ, Bautista, DS and Graham, FL (1 988) Virology 61 4-61 7) or after linearization and cotransformation into a recombination competent E. co / i strain (Chartier, C, Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A. and Mehtali, M. (1 996) J. Virol 70: 4805-481 0). Both methods are relatively complex due to inherent limitations: when recombined in 293 cells the problem is that unwanted.
  • Recombinant or wild-type viruses can arise. Therefore, a clonal isolation of the recombinant viruses by plaque assay on 293 cells and a thorough analysis of the isolated rAd is required before the multiplication.
  • the problem with recombination in E. coli is that the recombination-competent bacterial strain provides very low plasmid yields, which makes analysis of the recombined plasmids more difficult, since the transformation of an E. coli strain with a higher plasmid yield is first necessary.
  • Newer, but not yet widely used methods for rAd construction are based on the insertion of foreign DNA into the context of the adenovirus genome by direct ligation.
  • One method is based on ligation of a fragment of the (manipulated) 5 ' viral end with a fragment which contains the rest of the viral genome, followed by transfection of the ligation products into 293 cells (Mizuguchi, H. and Kay, MA (1998 ) Hum. Gene Ther. 9: 2577-2583).
  • Another method is based on the use of the cosmid cloning technique.
  • cosmid vectors are used which contain the E1-deleted adenovirus genome and a polylinker with unique restriction sites for the insertion of foreign DNA.
  • linearized cosmid vector and foreign DNA to be inserted are packaged in vitro in lambda phage heads. After infection of E. coli, circular cosmids develop, from which linear rAd genomes can be released by restriction digestion and which are then transfected into 293 cells (Fu, S. and Deisseroth, AB (1997) Hum. Gene Ther. 8: 1321 -1330).
  • the cloned vector genomes either linearly with terminal inverted terminal repeats (ITRs) or in the circular plasmid with a ⁇ ea / -to-ta / 7 configuration of the ITRs are available.
  • ITRs terminal inverted terminal repeats
  • cloned Vector genomes differ structurally from natural adenovirus genomes, which contain a covalently bound viral protein (terminal protein, TP) on both ITRs.
  • pTP viral preterminal protein
  • the pTP is then processed by a protease to form the TP, which in the next round of replication - alongside the ITRs - is an important part of the substrate that is recognized by the replication machinery.
  • Viral genomes without TP are recognized about 1000 times worse than naturally replicated viral genomes with TP (overview in: Hay, RT, Freeman, A., Leith, I., Monoghan, A. and Webster, A. (1 995) Curr. Top. Microbiol Immunol. 1 99: 31-48).
  • the first replication of a cloned rAd vector genome without TP is therefore a rare event (approx. 1 0-100 events per 1 0 6 293 cells transfected). For this reason, the methods described above are only suitable for obtaining clonal populations of rAd. However, they are not suitable for generating complex populations of rAd that would require an efficient conversion of a complex mixture of cloned vector genomes into a complex mixture of replicated rAd.
  • Donor plasmid with 5'ITR complete packaging signal, foreign DNA and single loxP recognition sequence and two
  • Hardy et al. (1 997) can only be achieved by contransfection of virus DNA together with the donorplamide. Deproteinized viral DNA was used for this, which therefore has no terminal protein (TP).
  • the donor virus substrate introduced is therefore different from the infectious donor virus genomes with TP which are introduced by infection in the present invention.
  • An essential advantage of the present invention is that such natural substrates are available for adenoviral replication.
  • the introduction of the donor virus genome by infection was by Hardy et al. (1 997) also tried, but the contamination with donor viruses was so high in the first round of propagation that this was not investigated further.
  • the difference to the high purities of the invention is due to the disadvantage of the donor quarters due to the deleted packaging signal.
  • the construction of complex ad populations was developed by Hardy et al. neither examined nor discussed.
  • An object of the invention was therefore to provide a system for simple
  • Another task was to provide a system with which even complex recombinant adenoviruses can be generated.
  • a system for producing recombinant adenoviruses comprising (a) at least one donor virus with an at least partially deleted viral packaging signal which is framed by two recognition sites for a site-specific recombinase, (b) a packaging cell line that expresses the site-specific recombinase and
  • the novel method for rAD production according to the invention has decisive advantages over the methods described so far.
  • complex mixed rAd populations can be generated, which was not possible according to the prior art.
  • this creates the prerequisites for the construction of gene banks in an adenoviral context.
  • the essence of the new system according to the invention is that the need to convert cloned vector genomes into infectious replicated vector genomes is avoided by generating the rAd directly by enzymatic site-specific insertion of foreign DNA into a replicating virus.
  • a site-specific recombinase is used, for example recombinases from the Int family, such as Cre recombinase or Flp recombinase.
  • the reactions catalyzed by these recombinases depend on the topology of the recognition sites: If two recognition sequences lie in parallel orientation on the same DNA molecule, these site-specific recombinases catalyze the excision of the region in between as a circular molecule, with a single recognition sequence remaining at the excision site. This reaction is reversible, but the equilibrium lies on the excision side (excision / insertion reaction) for thermodynamic reasons.
  • Adenoviral packaging signals contain repeated functionally additive sequence motifs, to which cellular factors that have not yet been characterized specifically bind. Binding of these factors is necessary for efficient packaging of the replicated viral genomes in the viral envelopes.
  • the packaging signal of human adenovirus serotype 5 is currently best characterized: If one or more of the repeated, functionally additive sequence motifs ("A repeats") are deleted in the packaging signal, the partially deleted packaging signal ( ⁇ ) obtained in this way results in reduced packaging efficiency and thus reduced virus growth (Schmid, Sl and Hearing, P. (1 997) J. Virol. 71: 3375-3384).
  • the cellular factors also represent a limiting substrate, so that when a virus with a complete packaging signal is present at the same time, the growth reduction of a virus with a partially deleted packaging signal is additionally increased (Imler, JL, Bout, A., Dreyer, D., Diederle, A., Schultz, H., Valerio, D., Mehtali, M. and Pavirani, A. (1 995J Hum. Gene Ther. 6: 71 1 -721).
  • Recombinase is framed.
  • Packing signal optionally two detection points for contains a rarely cutting restriction endonuclease (in particular a restriction endonuclease with a recognition sequence> 8 bp, preferably> 10 bp) and (iv) insertion sites for foreign DNA or inserted foreign DNA. , ...
  • the packaging cell line is first infected with the donor virus.
  • the partially deleted packaging signal of the donor virus is cut out by the corresponding recognition sites in the donor virus genome by the site-specific recombinase expressed by the packaging cell line.
  • a high level of expression of the site-specific recombinase is required.
  • the donor plasmid with the transgene cassette to be inserted or the complex donor plasmid population with a large number of sequences in the context of the donor plasmid is introduced into the cells by transfection.
  • Different types of donor plasmids which differ slightly in their structure, then lead to the formation of the rAd by site-specific insertion (excision / insertion or terminal exchange, see below and Figure 2) via likewise slightly different reactions.
  • the donor plasmid or parts thereof with the transgene cassette or the gene bank and the complete viral packaging signal are inserted site-specifically into the insertion site of the donor virus ⁇ acceptor substrate via the recognition site (s) for the site-specific recombinase.
  • the resulting rAds contain the transgene cassette or the gene bank and the complete viral packaging signal.
  • they contain the covalently bound TP on one or both ITRs, which is why a single insertion event to produce a infectious and normally replicating rAd leads.
  • a complex mixture of donor plasmids therefore leads to the formation of an equally complex mixture of rAd.
  • Type 1 donor plasmids contain a bacterial backbone with a bacterial resistance gene and a bacterial origin of replication, the complete viral packaging signal, followed by a polylinker for inserting foreign DNA or already inserted foreign DNA, or - framed by a promoter and a polyadenylation signal - a polylinker for inserting coding sequences or already inserted coding sequences and a recognition site for the site-specific recombinase located in front of the viral packaging signal.
  • the complete donor plasmid is inserted into the insertion site of the donor virus ⁇ acceptor substrate by the site-specific recombinase via an insertion / excision equilibrium reaction.
  • the resulting rAd contain two recognition sites for the site-specific recombinase (see Figure 2A).
  • Type 2 donor plasmids contain a bacterial backbone with a bacterial resistance gene and a bacterial origin of replication, the viral ITR and the complete viral packaging signal, followed by a polylinker for the insertion of foreign DNA or already inserted foreign DNA, or - framed by a promoter and a polyadenylation signal - a polylinker for inserting coding sequences or already inserted coding sequences and two cutting-edge reaction endonucleases with a more than 8 bp long recognition sequence that frames the bacterial backbone, one of the interfaces directly is next to the viral ITR.
  • the clonal or complex donor plasmid population is digested with the rarely cutting restriction endonuclease. This releases fragments which contain the viral ITR, the complete viral packaging signal, the inserted foreign DNA and a single recognition sequence for the site-specific recombinase in sequential order.
  • the longer the recognition sequence of the rarely cutting restriction endonuclease the lower the likelihood of the occurrence of a corresponding sequence in the transgene cassette or individual sequences in the gene bank which would interfere with the release of these fragments.
  • the fragments are inserted by the site-specific recombinase via a terminal exchange reaction into the insertion site of the donor virus ⁇ acceptor substrate.
  • the resulting rAd contain only one recognition site for the site-specific recombinase (see Figure 2B).
  • Type 3 donor plasmids included all elements of the type 1 donor plasmids and a second recognition site for the site-specific recombinase, which is localized such that (i) both recognition sites are oriented in parallel and (ii) both recognition sites frame the bacterial backbone with the origin of replication and the bacterial resistance gene.
  • the bacterial backbone is first cut out by the site-specific recombinase.
  • the product is a circular DNA molecule that contains the complete viral packaging signal, the foreign DNA to be inserted and a single recognition site for the site-specific recombinase. This is then inserted into the insertion site of the donor virus ⁇ acceptor substrate by the site-specific recombinase via an insertion / excision equilibrium reaction.
  • the resulting rAd contain two recognition sites for the site-specific recombinase (see Figure 2C).
  • rAd When using donor plasmids of types 1 and 3, rAd is formed in which the inserted DNA and thus also the complete viral packaging signal is framed by two recognition sites for the site-specific recombinase which are repeated in parallel. The rAd are therefore still the substrate for the excision / insertion equilibrium reaction of the site-specific recombinase. The entire DNA, including the packaging signal, is cut out by the excision. This rAd is therefore preferably propagated on cells which do not express the site-specific recombinase. The selection against contamination with unprocessed donor viruses is made here solely via the partially deleted packaging signal.
  • rAd is formed which contains only one recognition site for the site-specific recombinase. They are not a substrate for the excision / insertion reaction but for the terminal exchange reaction. This is not associated with the loss of the packaging signal. RAd generated in this way can therefore be propagated both on the packaging cell line that expresses the site-specific recombinase (selection against contamination with unprocessed donor viruses (i) via the excision of the packaging signal by the site-specific recombinase and (ii) via the partially deleted packaging signal), as well on cells that do not express them (selection against contamination with unprocessed donor viruses only via the partially deleted packaging signal).
  • Human or non-human adenoviruses are used as the basis for the construction of the donor viruses in order to generate correspondingly clonal or complex populations of recombinant human or non-human adenoviruses.
  • Human adenoviruses are preferably used, for example serotype 5 (Ad5).
  • donor viruses can be used in which one or more non-essential genes have been deleted.
  • One or more essential genes can also be deleted, which must then be made available in trans by the packaging cell line or by the producer cells.
  • the producer cells used to multiply the donor virus or the recombinant viruses derived therefrom are cells or cell lines which are permissive for the corresponding, possibly partially deleted recombinant virus, for example the E1 -complementing 293 cells for multiplying E1-deleted Ad5-derived donor viruses or the derived clonal or complex populations of recombinant adenoviruses.
  • the packaging cell line is obtained on the basis of the producer cell line by stable transfection of the gene for the site-specific recombinase.
  • the expression of the recombinase gene can be constitutive or be adjustable.
  • the recombinase gene can be a fusion gene from the recombinase gene and the sequences coding for a nuclear localization signal in order to increase the concentration of the recombinase in the cell nucleus.
  • Recombinases from the Int family are preferably used as site-specific recombinases, for example the Cre recombinase or the Flp recombinase.
  • coding sequences and elements which control their expression are generally used as transgene (s) in the donor plasmids.
  • the sequence to be expressed is preferably provided with a promoter which is either constitutively active or regulatable. Viral or cellular promoters or combinations of the two can be used as promoters.
  • the genomic sequence or the cDNA of a gene can be used, the product of which is missing in the disease to be treated, occurs in unphysiological amounts or is defective. You can also use part of a genomic sequence that spans a mutation in the target gene and can recombine it homologously.
  • various genes can be used that slow down the growth or kill the tumor cells - possibly in combination with pharmaceuticals or by immunostimulation. One or more of them may modified genes of the pathogenic organism against which immunization is to be achieved.
  • complex rAd populations are particularly preferred according to the invention.
  • populations of coding sequences mixed into the donor plasmids for example cDNA banks from human or animal tissues or cells, are used. This can be done, for example, with the aim of isolating new genes.
  • mixed populations of mutated sequences of this gene are used in the donor plasmids. This can be used, for example, to generate gene banks with variants of a protein (for example enzyme or antibody) with the aim of functional optimization of this protein.
  • the coding sequences are surrounded by elements that control their expression (promoters, polyadenylation signals).
  • elements that control their expression promoter, polyadenylation signals.
  • Another possible area of use for complex populations of rAd is to construct banks of non-coding or non-expressed sequences, for example for the characterization or optimization of binding sites of DNA-binding proteins or enzymes.
  • the isolation of new genes with the properties sought or the isolation of variants of a known gene with modified properties can be carried out as follows: First, the titer of infectious particles present in the complex rAd population certainly. Then, in order to produce the so-called master plates, producer cells in multi-titer plates are infected with a defined, low number of infectious particles per well. After the producer cells are completely infected, a freeze / thaw lysate of the master plates is produced. Due to the stability of rAd, the master plates can be frozen and stored. The released, increased viruses are present in the protrusion of the wells.
  • a clonal population is understood to mean a population in which the same foreign DNA is incorporated in all adenoviruses belonging to the population.
  • Foreign DNA is understood to mean any DNA that is not adenovirus DNA.
  • a complex population which is also referred to as a complex mixed population, comprises different adenoviruses, which differ in that they each contain different foreign DNA.
  • a complex recombinant adenovirus population preferably comprises at least two types of recombinant adenoviruses, each of which contains different foreign DNA, in particular at least 10 different types, most preferably at least 100 different types.
  • the packaging signal is partially deleted, so that an increase in the donor virus (without donor plasmid) in the packaging cell line is inhibited, reduced or / and impaired.
  • the desired rAd can be selectively amplified compared to the donor virus and thus selected.
  • the packaging signal in the donor virus is preferably deleted by at least 10%, in particular at least 20% and particularly preferably at least 30% and up to 100%, more preferably up to 90% and particularly preferably up to 70% (% here means the number of bases deleted based on the total base number of the packaging signal).
  • FIG. 1 shows the donor virus structure and formation of a donor virus ⁇ acceptor substrate in a packaging cell line which expresses the site-specific recombinase (gray boxes: viral inserted terminal repeat units (ITRs); black boxes: partially deleted packaging signal ( ⁇ ); white triangles: recognition sites for the site-specific recombinase (RS); white circles: viral terminal protein (TP)).
  • FIG. 2 shows the general structure of the preferred donor plasmids of type I (A), type II (B) and type III (C) and the principle of recombinant adenovirus production by site-specific recombination with the donor virus ⁇ v acceptor substrate in a packaging cell line, which the site-specific recombinase expressed (gray boxes: viral inserted terminal repeat units (ITRs); black boxes: complete viral packaging signal ( ⁇ ); white triangles: recognition sites for the site-specific recombinase (RS); white circles: viral terminal protein (TP); arrow: promoter (P); pA: polyadenylation signal; RCE: recognition site for a rarely cutting endonuclease).
  • ITRs viral inserted terminal repeat units
  • RS complete viral packaging signal
  • RS recognition sites for the site-specific recombinase
  • TP site-specific recombinase
  • arrow promoter
  • pA polyadenylation signal
  • RCE recognition site
  • Figure 3 shows a schematic overview of the use of adenovirus cDNA expression libraries for the identification of genes which induce a given phenotype in a functional cell-based assay.
  • FIG. 4 shows the genome structures of the donor viruses clantis and Ad / antisU, which are part of a system for the construction of clonal or complex populations of recombinant E1-deleted adenovirus serotype 5, and their functional characterization.
  • (4A) Schematic structure of Ad / antis ⁇ and Ad / antisW as well as the donor virus ⁇ acceptor substrate formed by Cre // ⁇ ° -mediated excision of the packaging signal and interfaces for Nhe I, which in the Analyzes were used in (4b) (gray boxes: viral inverted terminal repeat units (ITRs); black boxes with Latin numbers: so-called A repeats of the partially deleted packaging signal ( ⁇ ); white triangles: recognition sites for Cre recombinase (loxP); S : 929Bp spacer; gray boxes: Inverted terminal repeats of Ad5 (ITRs).
  • FIG. 5 shows the structures of the donor plasmids pCBI-3, pCBII-3, pCBIII-3, pCBI-CMVIl, pCBII-CMVIl and pCBIII-CMVIl, which are part of a system for the construction of clonal or complex populations of recombinant E1-deleted adenovirus serotype 5 are, as well as their • polylinker for the insertion of DNA (white circles: bacterial origin of replication (ori); Amp R: ampicillin resistance gene; gray boxes: 5'-inverted terminal repeat of Ad5 (5 'ITR); black boxes: complete packaging signal of Ad5 containing so-called A repeats l-VII ( ⁇ ); white triangles: recognition sites for Cre recombinase (
  • FIG. 6 shows the structure of the donor plasmids pCBI-DsRed, pCBII-DsRed and pCBIII-DsRed and the recombinant adenoviruses AdCBI-DsRed, AdCBII-DsRed and AdCBIII-DsRed formed from these donor plasmids by recombination with Ad / antis. Furthermore, the size of the Ps? AI fragments, in particular that of the 5 ' terminal Ps /? AI fragments, which were used in the analysis in FIG. 8A to distinguish between viral DNA from Adlantis and the newly formed recombinant adenoviruses.
  • the structures of the recombinant adenoviruses indicate the binding sites of the primers and the size of the corresponding PCR products, the formation of which in FIG. 8B confirms the formation of the recombinant adebnoviruses (round white circle (ori): bacterial origin of replication; white triangle (loxP): / oxP recognition site, ⁇ : complete packaging signal from Ad5; ⁇ *: partially deleted packaging signal from Ad5; black boxes: inverted terminal repetitions of Ad5 (ITRs); S: spacer; RSV: RSV promoter; bGHpA: bovine Growth hormone polyadenylation signal (bovine growth hormone polyadeny / ation signal); DsRed: open reading frame of the DsRed reporter gene).
  • round white circle (ori) bacterial origin of replication
  • white triangle (loxP) / oxP recognition site
  • ⁇ complete packaging signal from Ad5
  • ⁇ * partially deleted packaging signal from Ad5
  • black boxes inverted terminal repetitions
  • FIG. 7 shows the analysis of the mixtures of residual donor virus and newly formed recombinant adenoviruses obtained using the Ad / antisl donor virus and the pCBI-DsRed, pCBII-DsRed or pCBIII-DsRed donor plasmids.
  • 10 6 CIN 1004 cells were infected with 5 infectious particles Ad / antis ⁇ per cell and then transfected with 10 ⁇ g pCBI-DsRed, pCBII-DsRed (1-Scel-digested) or pCBIII-DsRed.
  • Three completely independent experiments were carried out for each donor plasmid.
  • FIG. 8 shows the analysis of the mixtures of residual donor virus and newly formed recombinant adenoviruses obtained at the level of the viral DNA when using the Adlantis donor virus and the pCBI-DsRed, pCBII-DsRed or pCBIII-DsRed donor plasmids.
  • (8A) shows the cleavage of 1 ⁇ g of the shepherd extracts with PshA ⁇ . Viral DNA from the Adlantis donor virus is applied as a control. This cleavage enables the 5 ' - terminal fragments of the newly formed recombinant adenoviruses and the donor virus Ad / antis ⁇ to be distinguished. (see Figure 6). When using pCBI-DsRed and pCBIII-DsRed as donor plasmid, only the 3909 bp 5 ' terminal fragment of Ad / antis ⁇ can be recognized.
  • both the 4581 bp 5 ' terminal fragment of AdCBII-DsRed and the 3909 bp 5 ' terminal fragment of Ad / ant / ' s ⁇ are in a ratio of about 1: 1 to recognize. This shows that the formation of AdCBII-DsRed from pCBII-DsRed is significantly more efficient than that of AdCBI-DsRed from pCBI-DsRed or that of AdCBIII-DsRed from pCBIII-DsRed.
  • (8B) shows the PCR detection of the DNA of the newly formed recombinant adenoviruses AdCBI-DsRed, AdCBII-DsRed or AdCBIII-DsRed in the shepherd extracts.
  • 1 ⁇ ⁇ of the shepherd extracts were used in a PCR with the specified primers AdCBI-s or bGHpA-s and Ad-as.
  • 1 ⁇ H 2 0 was used as a negative control. See FIG. 6 for the binding sites of the primers and the size of the corresponding PCR products (M: DNA size marker).
  • FIG. 9 shows the structure of the donor plasmids pCBII-DsRed and pCBII- / acZ and the recombinant adenoviruses AdCBII-DsRed and AdCBIIJacZ formed from these donor plasmids by recombination with the donor viruses Ad / antis and AdlantisW. Furthermore, the size of the Ps ⁇ AI fragments, in particular that of the 5 '-terminal s ⁇ AI fragments, which were used in the restriction analyzes in FIGS. 10 and 11 to differentiate between viral DNA of the donor viruses and the newly formed recombinant adenoviruses.
  • Figure 10 shows the experimental scheme that for the preparation of large preparations of the recombinant adenoviruses AdCBII-DsRed and AdCBIIJacZ was used. According to this scheme, 3 parallel independent experiments were carried out for both Dobnorviruses Ad / antis ⁇ and AdlantisW in combination with the donor plasmids pCBII-DsRed or pCBII- / acZ.
  • Figure 1 1 shows the analysis of the virus mixtures, which were obtained in the amplification round 1 (A1) according to the scheme of Figure 10.
  • 1 ml of the freezer / tau lysate A1 was used to infect 10 6 293 cells. After the cytopathic effect appeared, the replicated viral DNA was isolated by shepherd extraction. 1 ⁇ g of the shepherd extracts was then digested with PshA ⁇ .
  • This enzyme provides characteristic fragments from the 5 ' end of the donor viruses Adlantis ⁇ and AdlantisW as well as the recombinant adenoviruses AdCBII-DsRed and AdCBII- / acZ (see FIG. 9).
  • Control (C) was 1 ⁇ g of purified DNA from the donor virus Ad / antls ⁇ or AdfantisW used in each case.
  • the upper two figures show the results with pCBII-DsRed as donor plasmid (recombinant adenovirus AdCBII-DsRed), the two lower ones with pCBII- / acZ (recombinant adenovirus AdCBII- / acZ).
  • Adlantis ⁇ was used in the right-hand figures and AdlantisW in the left-hand figures as a donor virus.
  • the three independent experiments (a, b, c) with proliferation on 293 or CIN 1 004 cells are shown in the figures.
  • FIG. 12 shows the analysis of large preparations of the recombinant adenoviruses AdCBII-DsRed and AdCBII- / acZ, which were obtained according to the experimental scheme of FIG. 10.
  • the viral DNA was extracted from the purified infectious particles and 1 ⁇ g of the purified DNA was digested with PshA ⁇ .
  • This enzyme provides characteristic fragments from the 5 ' end of the donor viruses Ad / antis ⁇ and AdlantisW as well as the recombinant adenoviruses AdCBII-DsRed and AdCBII- / acZ (see FIG. 9).
  • Control (C) was 1 ⁇ g of purified DNA from the Adlantisl or AdlantisW donor virus used.
  • the upper two figures show the results with the recombinant adenovirus AdCBII-DsRed, the two lower ones with the recombinant adenovirus AdCBII- / acZ.
  • Ad / antis ⁇ was used in the right-hand figures and AdlantisW in the left-hand figures as a donor virus.
  • the three independent experiments (a, b, c) with proliferation on 293 or CIN 1 004 cells are shown in the figures.
  • FIG. 13 shows the determination of the titer of intact infectious particles and the total titer of viral particles in the large preparations of AdCBII-DsRed and AdCBII- / acZ, which were produced according to the scheme in FIG.
  • the titer of intact infectious particles was determined by dilution endpoint analysis on 293 cells (black bars), the total titer of viral particles by measuring the photometric absorption of the virus preparation (white bars). The mean of the three independent experiments and the standard deviation are shown. The ratio of the total titer of viral particles to the titer of infectious particles is shown above the pairs of bars.
  • FIG. 13 shows the determination of the titer of intact infectious particles and the total titer of viral particles in the large preparations of AdCBII-DsRed and AdCBII- / acZ, which were produced according to the scheme in FIG.
  • the titer of intact infectious particles was determined by dilution endpoint analysis on 293 cells (black bars
  • FIG. 15 shows the testing of the large preparations of AdCBII-DsRed and AdCBII- / acZ, which were prepared according to the scheme of FIG. 10, for contamination with replication-competent wild-type adenoviruses (RCA).
  • RCA replication-competent wild-type adenoviruses
  • FIG. 16 shows the high efficiency with which replication-competent wild-type adenoviruses (RCA) arise from Ad / antis ⁇ , but not from AdlantisW, after infection of CIN1004 cells.
  • 293 or CIN1004 cells were infected with 5 infectious particles per cell Ad / antis ⁇ or 1 infectious particles per cell AdlantisW. After the virus-induced cytopathic effect appeared, the cells were disrupted by freeze-thaw lysis and the lysates were tested for the presence of RCA by means of PCR. Primers were used which lead to the formation of a 600 bp product when RCA DNA is present.
  • H 2 0 or freeze / tau lysates of sham-infected 293 cells (mock) were used as negative controls, freeze / tau lysates of RCA-infected cells (M: DNA size marker) as positive controls (PC).
  • FIG. 17 shows the determination of the number of independent recombinant adenovirus clones which arise when using the Adlant / sl or AdlantisW donor viruses and type 2 donor plasmids from 10 6 CIN 1 004 cells.
  • 1 0 6 CIN 1 004 cells were infected with 5 infectious particles per cell Ad / ant / s ⁇ (top) or 1 infectious particles per cell AdlantisW (bottom) and then each with 12 vg of different mixtures of / -Scel-digested pCBII-DsRed and pCBII- / acZ transfected. Mixing ratios from 50: 1 to 500,000: 1 were used.
  • the Huh7 cells were stained with XG al.
  • the number of pC BI I- / acZ resulting en / acZ-transducing units could then be determined by counting the blue-stained cells.
  • the bars indicate the mean of the total number of LTU and the standard deviation in those experiments in which blue cells could be detected. The ratio of the number of positive experiments to the total number of experiments is shown above the bar.
  • FIG. 18 shows the experimental scheme for the production of adenoviral cDNA expression banks and their use for the identification of genes which produce a certain phenotype in a test system (white circle: bacterial origin of replication (ori); white arrow: ampicillin resistance gene (amp); white triangle : / oxP recognition site (loxP); black boxes: terminal inverted repeats of Ad5 (ITRs); ⁇ : complete packaging signal of Ad5; Ad5 ⁇ E1 ⁇ E3: coding sequences of Ad5 with deletion of the E1 and E3 region; boxes with arrow: CMV Promoter (CMV); pA: CMV polyadenylation signal).
  • CMV CMV Promoter
  • FIG. 19 summarizes the experimental procedure for the construction of the expression library for human liver cDNA in the donor plasmid pCBII-CMVII (white circle: bacterial origin of replication (ori); white arrow: ampicillin resistance gene (amp); white triangle: / oxP recognition site oxP ); black boxes: 5 ' terminals inverted repeat of Ad5 (5'ITR); ⁇ : full packaging signal from Ad5; Box with arrow: CMV promoter (CMV); pA: CMV polyadenylation signal.
  • FIG. 20 shows the characterization of the expression library for human liver cDNA in the donor plasmid pCBII-CMVIl (pCBII-CMVIl-LIVERcDNA), which had been prepared according to the scheme from FIG.
  • (20A) shows the determination of the size range of the inserted cDNAs.
  • 1 ⁇ g plasmid DNA from individual clones was digested with St7aBI.
  • pCBII-CMVII was digested with SnaB ⁇ without inserted foreign DNA.
  • This enzyme supplies a 3554 bp fragment from the plasmid backbone, as well as a further fragment which contains the expression cassette together with the CMV promoter, inserted cDNA and CMV polyadenylation signal (see FIG. 19).
  • the size of this fragment can be estimated by subtracting the sum of the sizes of the CMV promoter and the polyadenylation signal (632 bp) from the size of the inserted cDNA.
  • (20B) shows the presence of the cDNAs for hAAT (top) and hFlX (bottom) using PCR.
  • the binding sites of the primers used and the size of the products are shown schematically.
  • 50, 200 or 500 ⁇ g of the plasmid bank were used in the PCR.
  • H 2 0 and 10 ng pCBII-CMVII served as negative controls
  • 10 ng each of a plasmid with the complete reading frame of hAAT (top) or hFlX (bottom) served as positive controls (PC).
  • FIG. 21 shows the experimental scheme that was used in the conversion of the expression library for human liver cDNA in the donor plasmid pCBII-CMVIl (“pCBII-CMVIl-LIVERcDNA”) into adenoviral cDNA expression banks.
  • Figure 22 shows the controls for the complexity and efficiency of the V u r u s n t h e n g e d u r E d i n e n u n g e n e n u n g e n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n g n e n u n e n g u n e n
  • Non-infected and non-transfected Huh7 cells (ni / nt) served as negative controls, Huh7 cells, which had been infected with 20 infectious particles per cell of a recombinant adenovirus with an RSV promoter-driven / acZ expression cassette, served as negative controls ( AdRSV- / lacZ)
  • FIG. 23 shows the characterization of the ad enoviral liver cDNA expression banks Ad / a / 7f / sLIVERcDNAI & II with respect to the sizes of the inserted cDNAs.
  • Individual virus clones isolated by piaque assay on 293 cells were used to infect 10 6 293 cells each. After 36 h, the replicated viral DNA was extracted and subjected to a restriction analysis with PshA ⁇ . This enzyme provides a characteristic fragment from the 5 'end of the recombinant adenovirus, the size of which is composed of 3667 bbp vector sequences and the size of the inserted cDNA.
  • FIG. 24 shows the extent of the contamination of the adenoviral liver cDNA expression banks Ad / a / 7 / sLIVERcDNAI & II with replication-competent adenoviruses (RCA). 10 7 Huh7 cells were infected with 1 - 10 8 infectious particles (IP) from the expression banks.
  • IP infectious particles
  • FIG. 25 shows in table form the characterization of the inserted cDNAs in the clones I-6, I-8, I-9, 1-1 5, 1-1 7 isolated from the adenoviral liver cDNA expression bank Ad / a ⁇ t / sLIVERcDNA I by piaque assay and 1-1 8.
  • FIG. 26 shows in tabular form the characterization of the inserted cDNAs in the clones 1-1 9, I-24, I-25, I-26 and I-27 isolated from the adenoviral liver cDNA expression bank Ad / a ⁇ f / sLIVERcDNA I by piaque assay.
  • FIG. 27 shows the scheme on which the first screening round of the adenoviral liver cDNA expression banks (Ad / anf / sLIVERcDNA) was based for recombinant adenoviruses which contain the hAAT or hFlX cDNA.
  • adenoviral liver cDNA expression banks Ad / anf / sLIVERcDNA
  • 3 ⁇ 10 3 293 cells were sown in 96-we // plates.
  • wells A1-F1 2 were then infected with 50 (first screening round hAAT) or 500 (first screening round hFlX) infectious particles per well.
  • Non-infected cells (wells G 1 -G6) and cells infected with 50 (first screening round hAAT) or 500 (first screening round hFlX) Ad / ant / s per well (wells G7-G 1 2) served as controls. After 7 days, the propagated viruses were released by freeze / thaw lysis of the cells in the master plates. In each case 40 ⁇ ⁇ of the virus-containing supernatants were used for the infection of 96-we // plates with 3 ⁇ 1 0 4 293 cells per well (master plates S1 A2). After the virus-induced cytopathic effect appeared after about 3 days, the cell culture supernatants were tested for hAAT or hFlX by ELISA.
  • Non-infected cells (wells G 1 -G6) and adfantisl particles infected with 1 (second screening round hAAT) or 10 (second screening round hFlX) per well (wells G7-G 1 2) served as controls.
  • the propagated viruses were released by freeze / thaw lysis of the cells in the master plates.
  • 40 ⁇ l of the virus-containing supernatants were used to infect 96-well plates with 3 ⁇ 1 0 4 293 cells per well (master plates S2A2).
  • the second screening round hFlX the cell culture supernatants of these master plates were tested for hFlX by ELISA after the virus-induced cytopathic effect (CPE) had occurred.
  • CPE virus-induced cytopathic effect
  • FIG. 29 shows the experimental scheme for the clonal isolation of recombinant adenoviruses which contain the hAAT or hFlX cDNA from the positive wells in S2A3 (second screening round hAAT) and S2A2 (second screening round hFlX).
  • Individual virus plaques are obtained by piaque assays with serial dilutions of the freeze / thaw lysates from the positive wells of the second round of screening. The plaque isolates are then individually expanded on 293 cells and the cell culture supernatants are tested for hAAT or hFlX by ELISA. Both positive PIaqueisolaten the presence of the hAAT or hFlX cDNA is then verified by sequencing.
  • FIG. 30 shows the results of the first screening round of the two adenoviral liver cDNA expression banks Ad / a / 7f / sLIVERcDNAI (top) and Ad / ant sLIVERcDNAII (bottom) for recombinant adenoviruses which contain the hAAT cDNA.
  • the raw data of the hAAT ELISA (OD 490 ) are shown with the supernatants of each of the 3 (a, b, c) master plates S1 A2, which were produced according to the scheme of FIG.
  • FIG. 31 shows the results of the second screening round of the adenoviral liver cDNA expression bank Ad / a / 7f / sLIVERcDNAI for recombinant adenoviruses which contain the hAAT cDNA.
  • the raw data of the hAAT-ELISA (OD 490 ) are shown with the supernatants of each 1 master plate S2A3, per selected positive subpopulation (1 -a-B9, 1 -a-D1, 1 -b-D10 and 1 -C-B8 ) the master plates S1 A2 were produced according to the scheme of FIG.
  • FIG. 32 shows the results of the first screening round of the adenoviral liver cDNA expression bank Ad / at7t / sLIVERcDNAI for recombinant adenoviruses which contain the hFlX cDNA.
  • the raw data of the hFlX-ELISA (OD 490 ) with the supernatants of the 9 (af) master plates S1 A2 are shown, which were produced according to the scheme of FIG.
  • FIG. 34 shows the results of the second screening round of the adenoviral liver cDNA expression bank Ad / a ⁇ r / sLIVERcDNAI for recombinant adenoviruses which contain the hFlX cDNA.
  • the raw data of the hFlX-ELISA (OD 490 ) are shown with the supernatants of the two master plates (A, B) S2A2, the per selected positive subpopulation (la-A1 1 and 1 -b-F5) of the master plates S1 A2 according to the scheme of Figure 289 with 10 infectious particles per well in S2A1 (A1 -F1 2: samples; G 1 -G6: negative controls 1 (supernatants of uninfected 293 cells; G7-G 1 2: negative controls 2 (supernatants with Ad / antisl infected cells); F1 - F9: standard series hFlX (1: 2 dilution levels, starting with 25 ng hFlX / ⁇ l); F1 0-F1 2: blank value).
  • the system according to the invention for the production of rAd was implemented for the construction of clonal or complex populations of recombinant E1-deleted human adenoviruses of serotype 5 (Ad5).
  • the packaging signal of Ad5 consists of seven so-called A repeats, which are between nt 200 and nt 380 at the 5 'end of the Ad5 genome (Schmid, Sl and Hearing, P. (1 997) J. Virol. 71: 3375-3384) ,
  • the Cre // oxP recombination system of bacteriophage P1 was used as the site-specific recombination system, consisting of the Cre recombinase and the / ox sequence recognized by it (Sternberg, N.
  • Ad5-derived E 1 -deleted replication-deficient viruses are used as donor viruses, the packaging signal of which (i) is partially deleted and (ii) is framed by parallel / oxf sequences.
  • the donor viruses also have a 2.7 kbp deletion in the E3 region and can therefore take up to 8 kbp foreign DNA.
  • Adlantis ⁇ the A repats VI and VII have been deleted, so it contains the A repeats l-V (nt 1 94-358 of the Ad5 genome).
  • AdlantisW the A repeats III, IV and V are deleted, so it contains the A repeats I, II, VI and VII (nt 1 94-271 and then nt 355-542 of the Ad5 genome).
  • the donor virus genomes were constructed by homologous recombination in E. coli. First, S / ji / tt / e plasmids were constructed which contained the 5 'end of the donor viruses (pAd2lis for Ad / antis ⁇ and pAd2lis ⁇ for AdlantisW).
  • the starting plasmid for the construction of pAd2lis was p_E1-2lox, which, in sequential order, the 5 'ITR of Ad5, an / ox sequence, a partially deleted packaging signal from Ad5 with the repeats IV ( ⁇ IV-VII), and a 929 bp size was not -coding spacer fragment, a second parallel / ox sequence and then the nt 3524-5790 of the Ad5 genome (Hillgenberg, M., Schnieders, F., Loser, P. and Strauss, M. (2001) Hum Gene Ther. 1 2: 642-657).
  • the functional elements mentioned were released from p_E1-2lox as a 3008 bp / 4 / 7lll // 3s ⁇ II fragment and inserted into the S ⁇ i / r ⁇ / e plasmid pHVAd2 (Sandig, V., unpublished) via the same restriction sites, from which pAd2lis emerged.
  • the partially deleted packaging signal ⁇ VI-VII in pAd2lis was replaced by the partially deleted packaging signal ⁇ III-V.
  • the starting point was the plasmid pSLITRPS, which contains the first 542 bp of the Ad5 genome including the 5 'ITR and the complete packaging signal (Hillgenberg, M., Schnieders, F., Lenter, P. and Strauss, M. (2001) Hum. Gene Ther. 1 2: 642-657).
  • a 704 bp Sa / l / ⁇ / ⁇ l fragment was cleaved, which contained the Ad5 sequences mentioned and was inserted into the Dsal site of the plasmid pBSSK- (Stratagene), resulting in plasmid pBSITRPS.
  • an 84 bp Dsal / M / t / NI fragment was cleaved, which corresponds to nt 272-355 of the Ad5 genome and which contains repeats III-V.
  • the vector was obtained by religation of the plasmid pBSITRPS ⁇ , which contains the partially deleted packaging signal ⁇ III-V.
  • pHVAd 1 (Sandig, V., unpublished) contains the rest of the Ad5 genome with a 2.7 kbp deletion in the E3 region.
  • the genomes of the donor viruses Adlantis ⁇ and AdlantisW were released from the plasmids pAdl lis and pAdl lis ⁇ obtained by this recombination by digestion with Pac ⁇ and then transfected into 293 cells.
  • the 293 cells complement the E1 deficiency of the donor virus, resulting in a Virus propagation can take place.
  • the infectious viruses obtained from this were then propagated to 293 cells.
  • Ad / antis ⁇ was released as a supernatant from thoroughly infected lysed 293 cells and then purified using CsCI density gradients, AdlantisW was used directly as a supernatant from thoroughly infected lysed 293 cells.
  • the cell line derived from 293 cells comes as the packaging cell line
  • CIN1004 for use, which constitutively expresses the gene for a core-localized Cre recombinase (Hillgenberg, M., Schnieders, F., Lenderr, P. and Strauss, M. (2001) Hum. Gene Ther. 12: 642- 657).
  • This cell line could be obtained by using a bicistronic vector in which the expression of a nuclear-localized Cre recombinase was linked to that of the selectable neo gene via an internal ribosome entry site. After transfection of 293 cells with this vector, a direct selection for high expression of the Cre recombinase could be made via a selection for the high expression of the neo gene.
  • Donor plasmids are used, each corresponding to type 1, 2 and 3 donor plasmids (pCBI, pCBII and pCBIII). They contain one (pCBI, pCBII) or two (pCBIII) / oxP recognition sites and the complete packaging signal of Ad5 (A repeats l-VII, nt 194-526 of the Ad5 genome). pCBII also contains two recognition sites for the rarely cutting restriction endonuclease I-Scel (18 bp recognition sequence).
  • the plasmids are each in various forms (see Figure 54), for example with a polylinker into which complete expression cassettes with promoter, coding region and polyadenylation signal can be inserted (pCBI-3, pCBII-3, pCBIII-3) or with a polylinker , which is framed by the hCMV promoter and the hCMV polyadenylation signal, for the insertion of coding sequences, for example transgenes or cDNA banks (pCBI-CMVIl, pCBII-CMVIl, pCBIII-CMVIl).
  • the donor plasmids were constructed on the basis of pMV, a plasmid which, in addition to a bacterial origin of replication (ColEI), a cos signal and the ampicillin resistance gene, is an I-Scel recognition site in sequence, nt 1-542 of the Ad5 genome (5 ' ITR and complete packaging signal), a polylinker containing 3 'ITR of Ad5 and a second I-Scel recognition site (Hillgenberg, M., Schnieders, F., Lenter, P. and Strauss, M. (2001) Hum. Gene Ther. 12: 642-657).
  • pCBI-3 and pCBI-CMVIl were first carried out by inserting a 107 bp-Xmal fragment containing a / ⁇ xP recognition sequence into the SprAI located in pMV between the Ad5-5 ' ITR and the Ad5 packaging signal Interface pMVI obtained.
  • a 905 bp fragment was released from pMVI, which contained the I-Scel recognition sequence, the Ad5-5 'ITR, the / oxf recognition sequence, the Ad5 packaging signal and the polylinker.
  • PCBI-3 was obtained by cutting out the part of the F1 replication origin as a 284 bp / o; oMI fragment and religation of the vector. By inserting a 688 bp fragment containing the hCMV promoter and the hCMV polyadenylation signal with an intermediate polylinker between the Pml ⁇ and / ael sites of the polylinker of pCBI-3, pCBI-CMV was obtained.
  • pCBII-CMV was obtained.
  • pClII was obtained.
  • pCBIII-3 and pCBIII-CMVII were constructed from pCBI-3 (see above).
  • the plasmid pCBIII-3 was first obtained by inserting a 107 bp fragment with a / ox sequence into the ⁇ / groMI site of the polylinker of pCBI-3.
  • pCBIII-CMV was obtained.
  • CIN 1004 cells were infected with Ad / antis or AdlantisW. After the virus-induced cythopathic effect appeared, the replicated viral DNA was isolated and subjected to a restriction analysis in which a distinction can be made between unprocessed donor virus and processed donor virus ⁇ -acceptor substrate. The fragment pattern corresponded to completely processed donor virus ⁇ acceptor substrate (FIG. 4B).
  • the comparison of the number per cell produced as progeny of infectious particles after infection of CIN 1 004 or 293 cells showed an approximately 100-fold reduction in growth of the donor viruses Adlantis ⁇ and AdlantisW on CIN 1004 cells (FIG.
  • a constitutive expression cassette for the reporter gene DsRed was inserted into the polylinker of the donor plasmids pCBI, pCBI I and pCBII I.
  • donor plasmids pCBI-DsRed, pCBII-DsRed and pCBIII-DsRed as well as the recombinant adenoviruses resulting from these donor plasmids by Cre / tox -mediated recombination with the donor virus ⁇ acceptor substrate AdCBI-DsRed, AdCBII-DsRed and AdCBIII-DsRed are shown in Figure 6.
  • pCBI-DsRed, pCBII-DsRed (digested with I-Scel) and pCBIII-DsRed were transfected into CIN 1004 cells which had previously been infected with Adlantis ⁇ .
  • the virus-induced cythopathic effect (CPE) appeared, the cells were lysed (freeze / thaw lysate amplification round 0, AO).
  • the virus-containing lysate obtained in this way was used to multiply the recombinant adenoviruses for the infection of 293 cells, which in turn were lysed after the appearance of the CPE (freezer / tau lysate amplification round 1, A1).
  • the total amount of the recombinant adenoviruses AdCBI-DsRed, AdCBII-DsRed and AdCBIII-DsRed contained in AO and A1 were then determined.
  • DsRed transducing units DsRed transducing units
  • the total amount of infectious particles in A0 and A1 was titrated by dilution endpoint analysis on 293 cells (FIG. 7).
  • DTUs were detected and recombined adenoviruses were formed.
  • the total amount of DTU when using the donor plasmids pCBI-DsRed and pCBIII-DsRed was about 100 in A0 and about 1000 in A1.
  • the total amount of infectious particles was about 10 5 in AO and about 10 7 in A1, which indicated that the recombinant adenoviruses were heavily contaminated with residual donor virus.
  • the total amount of DTU was significantly higher at about 10 5 in AO and about 10 7 in A1 for a comparable total amount of infectious particles.
  • PCR analyzes were carried out with the same herbal extracts, in which primer pairs were used which were only from the recombinant adenoviruses AdCBI-DsRed, AdCBII-DsRed and AdCBIII-DsRed, but not from the donor virus or Donor plasmids deliver a product (see Figure 6).
  • the product characteristic of the recombinant adenoviruses occurred in all experiments (FIG. 8B).
  • donor plasmids of type 2 (derivatives of pCBII-3 or pCBII-CMVII, see FIG. 5) were subsequently used to generate clonal and complex populations of recombinant adenoviruses.
  • the donor plasmids pCBII-DsRed and pCBII- / acZ were used, which contain constitutive expression units driven by the RSV promoter for the reporter genes DsRed and lacZ as transgenes.
  • pCBII-DsRed see above
  • pCBII- / acZ was obtained by inserting the expression cassette into the polylinker of pCBII-3.
  • Both plasmids and the recombinant adenoviruses AdCBII-DsRed and AdCBII- / acZ resulting from recombination with the donor virus ⁇ acceptor substrate are shown in FIG. 9.
  • the efficient generation of recombinant adenoviruses from donor plasmids of type 2 in conjunction with Adlantisl as donor virus had already been shown in the previous experiments. Subsequently, it was tested under which conditions large preparations of clonal recombinant adenoviruses with sufficient purity can be obtained.
  • the system according to the invention for the production of recombinant adenoviruses enables the use of two selection principles, which may also be combined, when using type 2 donor plasmids Reduction of contamination by residual donor virus: (1)
  • the donor viruses have a deletion in the packaging signal.
  • the recombinant adenoviruses should therefore have a growth advantage. This advantage should be inversely related to the extent of deletion of the packaging signal, which is different in the Adlantisl and AdlantisW donor viruses.
  • CIN 1004 cells they are therefore not a substrate for the Cre // ox -mediated excision of the packaging signal, in contrast to the donor viruses whose packaging signal is framed by two / oxP sequences and whose growth on CIN 1004 cells is approximately 100 times is reduced (see Figure 4).
  • FIG. 10 summarizes the experimental procedure. After infection of 1 0 6 CIN 1 004 cells with Adlantisl or AdlantisW, the cells were transfected with the 1-Scel-digested donor plasmids pCBII-DsRed or pCBII- / acZ. After the virus-induced cytopathic effect appeared, the cells were lysed.
  • Each 1/5 ml of the virus-containing lysate thus obtained was used for propagation on 293 or CIN 1 004 cells, sequentially a 60 mm dish (amplification round 1, A1), a 1 50 mm dish (amplification round 2, A2) and finally 10 1 50 mm dishes (amplification round 3, A3) were used.
  • the viruses released from the last round of amplification were purified by means of CsCI density gradient centrifugation and, after separation of the CsCI by gel filtration, 2 ml of purified virus preparations were obtained.
  • the viral DNA was extracted from the purified virus preparations and again analyzed by cleavage with PshA. Only the characteristic 5 ' -terminal fragment of the recombinant adenovirus was recognizable in all purified virus DNAs (FIG. 1 2). Contamination with residual donor virus could therefore not be detected using this method. In addition, these results showed that the structure of the recombinant viruses had remained intact during the propagation.
  • the titer of intact infectious particles (by dilution endpoint analysis on 293 cells) and the total titer of viral particles (by measuring the photometric absorption of the virus preparation) was then determined for all purified large-scale preparations of AdCBII-DsRed and AdCBII- / acZ ( Figure 1 3). Both the titer of intact infectious particles (10 10 - 10 1 1 per ml) and the ratios of the total titer of viral particles to the titer of infectious particles (1 5 - 43) are within the scope of what is also possible with other common methods of production and Propagation of recombinant adenoviruses is obtained.
  • AdCBII-DsRed and AdCBII- / ac were then checked for contamination with replication-competent wild-type adenoviruses (RCA). As is known, these arise with a frequency that cannot be neglected when multiplying recombinant adenoviruses on 293 cells. This is based on homologous recombination events between the 5 ' termini of the recombinant adenoviruses and the 43445 ' terminal Bp of Ad5 inserted into the genome of 293 cells (and also of the CIN1004 cells derived therefrom). A double crossover event creates wild-type viruses that are no longer deficient in E1.
  • AdCBII-DsRed and AdCBII- / acZ are identical when using both donor viruses, it was unlikely that the RCA would arise from these recombinant adenoviruses during their multiplication. Rather, it had to be assumed that they would arise with a high probability after infection of CIN 1004 cells in AO specifically when Adlantisl was used and would then grow with the proliferation of the recombinant adenoviruses. To verify this, Adlantisl and Ad / antisl were passaged once through 293 and CIN 1004 cells. The same conditions were used as for the A0 for the production of recombinant adenoviruses according to the scheme of FIG.
  • AdlantisW is used as a donor virus in conjunction with donor plasmids of type 2 (pCBII-3 derivatives)
  • reproducible reproductions can be obtained by directly increasing the recombinant adenoviruses generated in AO to 293 or CIN 1004 cells, which ( 1) contain the recombinant adenoviruses with intact genome structure in high titers, (2) have a residual donor virus contamination of less than 0.001% and (3) are not contaminated with RCA.
  • the method according to the invention represents an advance over previous methods for producing clonal populations of adenoviruses, since it requires significantly fewer work steps and is therefore faster and moreover easier to handle. It is also cheaper in terms of material costs.
  • Adlantisl and AdlantisW were used as donor viruses and mixtures of the donor plasmids pCBII-DsRed and pCBII- / acZ to determine the number of independent rAd formation events, which is a measure of the complexity to be achieved with the system in the production of mixed rAd populations ).
  • Adlantisl 5 infectious particles per cell
  • Adlantisll 1 infectious particle per cell
  • 1 0 6 CIN 1 004 cells each with 1 2 ⁇ g different mixtures of / -Scel-digested pCBII-DsRed and pCBil- / acZ transfected. Molar mixing ratios of 50: 1 to 500,000: 1 were used.
  • AdlantisW The complexity of 50,000 independent clones per 10 6 cells achieved with Adlantisl means that when using only 2 x 10 7 cells (corresponding to 20 subconfluent 60 mm dishes), a total complexity of 10 6 independent clones, which is necessary for the construction of gene banks, for example adenoviral cDNA- Expression banks, is sufficient.
  • AdlantisW is unsuitable as a donor virus for the construction of cDNA expression banks, since a complexity of 10 6 independent clones would require the use of 10 8 -10 9 CIN 1004 cells (corresponding to 200 - 2000 subconfluent 60 mm dishes). In addition, this would require the transfection of a total of 2.4-24 mg cDNA expression bank in the donor plasmid. The proliferation of cDNA expression banks in plasmids to such amounts is not possible without loss of complexity.
  • the diagram in FIG. 18 shows the experimental procedure for the construction of adenoviral expression banks and the method for isolating cDNAs from them by means of a biological test system.
  • cDNA is synthesized, which is then directed between the CMV promoter and the CMV polyadenylation signal into the polylinker of the Donorplasmids pCBII-CMVIl is inserted.
  • a cDNA expression bank in pCBII-CMVII is obtained from this.
  • adenoviral cDNA expression bank with 10 6 independent clones, a total of 20 60 mm cell culture dishes, each with 10 6 CIN 1004 cells, were infected with 5 infectious Adlantisl particles per cell and then transfected with 1 2 ⁇ g l-Scel-digested plasmid bank. By multiplying the viruses generated in this way and subsequently cleaning the viruses, a high-titer purified adenoviral cDNA expression bank is obtained.
  • master plates are produced by infection of 293 cells in multiwell plates.
  • One or more infectious particles from the adenoviral cDNA expression bank are used per well, as a result of which defined monoclonal or oligoclonal subpopulations are increased.
  • the master plates can be stored for a long time due to the stability of adenoviruses.
  • the supernatants in the wells of the master plates contain the increased infectious adenoviruses.
  • they contain the proteins which are encoded by the cDNAs contained in the respective adenovirus clones, since the CMV promoter leads to their expression in the infected 293 cells.
  • a direct detection of a desired protein in the lysates can therefore serve as a test system, for example an enzyme-linked immunosorbant assay (ELISA).
  • the lysates are used to infect cells in a cell-based test system, in which a phenotypic change caused by the expression of the cDNA in the cells can be detected.
  • the recombinant adenoviruses can be clonally isolated from 293 cells by piaque assay from those wells of the master plates, the supernatants of which deliver the signal in such test systems.
  • the cDNAs can then be characterized, for example, by sequencing.
  • an adenoviral cDNA expression bank was constructed on the basis of human liver mRNA. From this adenovirus clones were isolated, which isolated the cDNAs of human alpha-1-antitrypsin ( hAAT) and human blood coagulation factor IX (hFlX). ELISAs were used as a detection system to detect these secreted proteins in the supernatants of the master plates. These serum proteins expressed in the liver were selected because they are exemplary for a gene that is strongly expressed in the liver (hAAT, Serum concentration about 2 g / l) and a weakly expressed gene in the liver (hFlX, serum concentration about 4 mg / l).
  • plasmid DNA from isolated clones was subjected to a restriction analysis with St? ABI. This enzyme cleaves the entire expression cassette including the CMV promoter, cDNA and polyadenylation signal. The size of the inserted cDNA can be estimated from the size of the corresponding fragment (FIG. 20A).
  • This plasmid library was then used to obtain adenoviral liver cDNA expression banks.
  • the experimental procedure is summarized in FIG. 21. Twenty 60 mm cell culture dishes, each with 10 6 CIN 1004 cells, were infected with 5 infectious Adlantisl particles per cell and then transfected with 1 2 ⁇ g l-Scel-digested plasmid bank pCBII-CMVII-LIVERcDNA per dish. After the virus-induced cytopathic effect (CPE) appeared, the cells were disrupted by freeze / thaw lysis and the lysates from the twenty dishes were combined (primary adenoviral liver cDNA expression bank, amplification round 0, AO).
  • CPE virus-induced cytopathic effect
  • Half of the lysate from AO was used to multiply the expression bank to infect four subconfluent 1 50 mm cell culture dishes with CIN 1004 cells. After the CPE had appeared, the cells were freeze-thawed open-minded (amplification round 1, A1). Half of the lysate from A1 was then used to infect nine subconfluent 150 mm cell culture dishes with 293 cells. After the appearance of the CPE, the cells were sedimented and disrupted by freeze / thaw lysis (round of amplification 2, A2). The viruses released in this way were purified by CsCI density gradient centrifugation and, after separation of the CsCI, two ml of purified adenoviral liver cDNA expression bank were obtained.
  • the efficiency of virus generation in AO was therefore very high. Isolated blue cells were detected in all of the three controls for the complexity, which indicated that in each dish at least one recombinant adenovirus had arisen from the 1: 50,000 diluted pCBII- / acZ.
  • the complexity in AO was therefore at least 50,000 independent clones per 60 mm shell. When the primary adenoviral expression banks were obtained from twenty 60 mm shells in AO, a complexity of at least 1 0 6 independent adenovirus clones could be assumed.
  • the plasmid bank pCBII-CMVII-LIVERcDNA was thus converted with high efficiency into adenoviral liver cDNA expression banks with a complexity of at least 10 6 independent adenovirus clones in two independent experiments.
  • F / sLIVERcDNA II were within the scope of what was also achieved with the system according to the invention in the generation of clonal adenovirus populations (see above).
  • Individual clones of recombinant adenoviruses were then obtained from the two purified adenoviral expression banks for characterization of the insert size range by piaque assay on 293 cells.
  • the PIaqueisolaten were then infected 293 cells and then the replicated viral DNA isolated and subjected to a restriction analysis with PsbAI. This enzyme provides a characteristic fragment from the 5 ' end of the recombinant adenoviruses, from the size of which the size of the inserted cDNAs can be estimated.
  • Adlantisl Since the use of Adlantisl as a donor virus is associated with the risk of contamination of the virus preparations with replication-competent wild-type adenoviruses (RCA), the extent of contamination was determined for Ad / a ⁇ r / sLIVERcDNA I and Ad / anf / sLIVERcDNA II. This resulted in a contamination of ⁇ 1% with Ad / a / 7 / VsLIVERcDNAI and approximately 10% with Ad / ant / sLIVERcDNAII (FIG. 24). Due to the lower contamination with RCA, all subsequent experiments were carried out with Ad / a / 7f / sLIVERcDNA I.
  • the first step was to characterize individual plaque isolates from Ad / ar / rvsUVERcDNA I with regard to the inserted cDNAs in order to be able to make a statement about the percentage of complete cDNAs in the bank.
  • the PCR products were then cloned into the pBSKS polylinker.
  • the inserts were then sequenced with primers that bind to the T3 and T7 promoters on both sides of the insertion site of the PCR products in the plasmid vector.
  • BLASTN www.ncbi.gov
  • the sequences were compared with sequence databases. The results are summarized in a table in FIGS. 25 and 26.
  • the cDNAs were identified for 9 of the 11 plaque isolates. In two of the inserts (plaque isolates 1-15 and 1-19), apart from homologies to chromosomal regions, no matches could be found with known cDNAs. They may therefore represent genes not yet characterized.
  • the remaining nine inserts were five complete cDNAs (I-6, I-8, 1-11, 1-17, I-26) and four 5 ' truncated cDNAs (1- 18, I-24, I-25, I-28).
  • the cDNAs encoded in six cases for serum proteins that are synthesized in the liver (apolipoprotein A, complement component 4 binding protein, histidine-rich glycoprotein, vitronectin, 2 x haptoglobin) and in three cases for intracellular proteins of the liver (deoxyguanosine kinase, cytochrome P450 and the proteasomal modulatory subunit PSMD9).
  • serum proteins that are synthesized in the liver
  • apolipoprotein A complement component 4 binding protein
  • histidine-rich glycoprotein histidine-rich glycoprotein
  • vitronectin 2 x haptoglobin
  • intracellular proteins of the liver deoxyguanosine kinase, cytochrome P450 and the proteasomal modulatory subunit PSMD9
  • T / ' sLIVERcDNA I has thus shown that more than 50% of the cDNAs were complete, that 2/11 inserts correspond to genes which have hitherto not been characterized and that all clearly identifiable cDNAs code as expected for genes expressed in the liver.
  • Screening of the adenoviral liver cDNA expression banks For the screening for recombinant adenoviruses which contain the cDNAs for hAAT or hFlX, sandwich ELISAs with the supernatants of cells which were in 96-we // plates with subpopulations from the adenoviral liver cDNA expression banks had been infected.
  • FIGS. 27-29 For the isolation of recombinant adenoviruses which contain the cDNAs for hAAT or hFlX, the procedure summarized in FIGS. 27-29 was followed.
  • the supernatants of the master plates S1 A1 were used to infect a further 96-we // plate with 293 cells.
  • master plates S 1 A2 screening round 1 amplification round 2).
  • the supernatants of the master plates S 1 A2 were then tested for hAAT or hFlX by ELISA.
  • oligoclonal subpopulations in the Master plates S1 A2 can be identified which contain recombinant adenoviruses with the hAAT or hFlX cDNA.
  • the second round of screening ( Figure 28) was then about reducing the complexity of these subpopulations.
  • master plates S2A2 screening round 2, amplification round 2).
  • the supernatants from the S 1 A2 master plates were then either tested directly by hISA or hFlX by ELISA, or used again to infect another 96-well plate with 293 cells.
  • the master plates S2A3 were then obtained, which were then tested for hAAT or hFlX by ELISA.
  • low-complex subpopulations in the master plates S2A2 and S2A3 could be identified that contain recombinant adenoviruses with the hAAT or hFlX cDNA.
  • Their separation in clonal form can then be carried out according to the scheme in FIG. 29: piaque assay on 293 cells can then be used to obtain individual adenovirus clones from the positive wells of the master plates S2A2 or S2A3. These can then be multiplied individually on 293 cells.
  • the adenovirus clones which contain the cDNAs for hAAT and hFlX can then be identified by testing the cell culture supernatants by means of ELISA.
  • T / ' sLIVERcDNAI were then selected and the titer of infectious particles was determined (master plate S1A2 a well B9: ⁇ 3 x 10 8 IP / ml; master plate S1A2 a well D1: ⁇ 10 8 IP / ml; master plate S1A2b wellülO: ⁇ 10 8 IP / ml; master plate S1A2 c well B8: ⁇ 3 x 10 8 IP / ml).
  • master plate S1A2 a well B9 ⁇ 3 x 10 8 IP / ml
  • master plate S1A2 a well D1 ⁇ 10 8 IP / ml
  • master plate S1A2b wellülO ⁇ 10 8 IP / ml
  • master plate S1A2 c well B8 ⁇ 3 x 10 8 IP / ml
  • the results of the ELISAs with the supernatants of the four master plates S2A3 are shown in FIG. 31. 2-4 wells per master plate were positive, indicating successful isolation of recombinant adenoviruses containing the hAAT cDNA.
  • individual virus plaques can be obtained by piaque assay on 293 cells according to FIG.
  • the plaque isolates can then be individually expanded on 293 cells and the cell culture supernatants can be tested for verification by ELISA for hAAT or hFlX.
  • the presence of the hAAT or hFlX cDNA can be verified in the adenovirus clones by sequencing.
  • adenoviral cDNA expression banks could thus be produced starting from mRNA which correspond to the generally required criteria for cDNA expression banks: a complexity of approximately 1 0 6 independent clones, a high proportion of complete cDNAs (> 50%), and the presence of cDNAs even at low level expressed genes. Furthermore, it was shown that screening of the adenoviral cDNA expression banks generated in this way via master plates with low-complex subpopulations is suitable for isolating adenovirus clones with the desired properties. The system according to the invention for generating the adenoviral cDNA expression banks and the methods according to the invention for their screening therefore appear to be generally suitable for identifying genes which produce a detectable phenotype in a biological test system.
  • the invention thus relates to a novel system for the production of recombinant adenoviruses (rAd); Areas of application are in particular medicine, veterinary medicine, biotechnology, genetic engineering and functional genome analysis.
  • the content of the invention is a novel system for the production of rAd.
  • the rAd are generated by site-specific insertion of foreign DNA into an infectious replicating virus.
  • clonal rAd populations can be produced more quickly and easily compared to previous methods.
  • the new process also enables the production of complex mixed rAd populations.
  • the content of the invention is also the use of new method of rAd production for obtaining complex gene banks in the adenoviral context, for example cDNA expression banks.
  • the rAd obtained in this way can be used for the transfer and expression of genes in cells and for the transfer of genetic material in animals and humans with the aim of gene therapy and / or vaccination. Furthermore, the complex rAd populations (gene banks) obtained in this way can be used for isolating new genes and for functional modification or optimization of known genes.
  • the system for rAd production preferably consists of a donor virus, the packaging signal (i) of which has been partially deleted and (ii) is framed by parallel-oriented recognition sites for a site-specific recombinase, a packaging cell line which expresses the site-specific recombinase and
  • Donor plasmids which contain (i) one or two recognition sites for the site-specific recombinase, (ii) the complete viral packaging signal, (iii) optionally two recognition sites for a rarely cutting restriction endonuclease and (iv) insertion sites for foreign DNA or inserted foreign DNA ,

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Abstract

La présente invention concerne un nouveau système destiné à produire des adénovirus recombinés (rAd). Les domaines d'application de ce système sont la médecine, la médecine vétérinaire, la biotechnologie, la génétique et l'analyse génomique fonctionnelle. Le système de l'invention destiné à produire des rAd comprend de préférence: un virus donneur dont le signal d'encapsidation (i) est partiellement sujet à la délétion et (ii) qui est encadré par des sites de reconnaissance à orientation parallèle destiné à une recombinase site-spécifique; une lignée cellulaire d'encapsidation qui exprime la recombinase site-spécifique; et des plasmides donneurs qui comprennent (i) un ou deux sites de reconnaissance pour la recombinase site-spécifique, (ii) le signal d'encapsidation viral complet, (iii) éventuellement deux sites de reconnaissance pour une endonucléase de restriction à coupure peu fréquente, et (iv) des sites d'insertion pour de l'ADN étranger ou de l'ADN étranger inséré.
EP02764728A 2001-07-18 2002-07-18 Systeme destine a produire des populations clonales ou complexes d'adenovirus recombines et leur application Withdrawn EP1409700A2 (fr)

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CN108048483A (zh) * 2018-01-30 2018-05-18 中国疾病预防控制中心病毒病预防控制所 复制型重组腺病毒HAdV-5载体系统及其应用

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EP1427816A4 (fr) * 2001-09-18 2004-12-01 Clontech Lab Inc Procede de producteur dde vecteurs adenoviraux s'appuyant sur une recombinase specifique d'un site
US8709778B2 (en) * 2008-10-28 2014-04-29 Xavier Danthinne Method of adenoviral vector synthesis
JP6236393B2 (ja) * 2011-11-09 2017-11-22 セダーズ−シナイ メディカル センター 転写因子に基づくペースメーカー細胞の生成およびその使用方法
FR3040845B1 (fr) 2015-09-04 2018-05-18 Schneider Electric Industries Sas Methode d'adressage automatique dans une architecture de communication ethernet
CN112468320A (zh) * 2020-11-09 2021-03-09 江苏方天电力技术有限公司 一种基于新型智能融合终端的自动拓扑识别方法

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US6228646B1 (en) * 1996-03-07 2001-05-08 The Regents Of The University Of California Helper-free, totally defective adenovirus for gene therapy
JP2002538758A (ja) * 1998-02-17 2002-11-19 ジェンザイム・コーポレイション 偽アデノウイルスベクターの製造方法

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