WO1999002647A2 - Vecteurs de clonage pour preparer des adenovirus de type virus minimaux - Google Patents

Vecteurs de clonage pour preparer des adenovirus de type virus minimaux Download PDF

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Publication number
WO1999002647A2
WO1999002647A2 PCT/DE1998/001940 DE9801940W WO9902647A2 WO 1999002647 A2 WO1999002647 A2 WO 1999002647A2 DE 9801940 W DE9801940 W DE 9801940W WO 9902647 A2 WO9902647 A2 WO 9902647A2
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WIPO (PCT)
Prior art keywords
cloning vector
adenoviral
reporter gene
minimal
cloning
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PCT/DE1998/001940
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German (de)
English (en)
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WO1999002647A3 (fr
Inventor
Moritz Hillgenberg
Peter LÖSER
Frank Schnieders
Volker Sandig
Michael Strauss
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Hepavec Ag Für Gentherapie
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Priority claimed from DE19744768A external-priority patent/DE19744768C2/de
Application filed by Hepavec Ag Für Gentherapie filed Critical Hepavec Ag Für Gentherapie
Priority to EP98944984A priority Critical patent/EP1003895A2/fr
Priority to JP2000502148A priority patent/JP2001509375A/ja
Publication of WO1999002647A2 publication Critical patent/WO1999002647A2/fr
Publication of WO1999002647A3 publication Critical patent/WO1999002647A3/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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer

Definitions

  • the invention relates to cloning gates for the production of adenoviral minimal viruses. Areas of application of the invention are medicine and the pharmaceutical industry.
  • Adenoviral vectors are efficient gene transfer vehicles for somatic gene therapy.
  • the first generation vectors consist of the complete genome of the adenovirus serotype 5 (Ad5) with deletions in the E1 and E3 regions into which foreign genes can be inserted. These vectors are capable of independent propagation in suitable cell lines which provide the El functions. Due to the remaining viral coding sequences, such vectors have a maximum transport capacity of approx. 8 Kb Fre d-DNA. Residual expression of adenoviral genes appears to be largely responsible for the immunological elimination of transduced cells in vivo, and thus also for the temporal limitation of transgene expression and the corresponding therapeutic effects (Yang, Y. et al., 1994, Cellular immunity to viral antigens limits El- deleted for gene therapy, Proc. Natl. Acad. Sci. USA 91, 4401-4411).
  • a promising approach to increasing transport capacity while simultaneously reducing immunogenicity is the outsourcing of all coding adenoviral genome sections from the therapeutic vector (Chen, HH et al., 1997, Persistence in muscle of an adenoviral vector that lacks all viral genes, Proc. Natl. Acad.See. USA 94, 1645-50).
  • adenoviral minimal viruses which, in addition to the therapeutic foreign gene to be transported, only the short signal sequences for the viral replication machinery (inverted terminal replication sequences, English inverted terminal repeats, ITRs) and for packaging in viral envelopes (English packaging signal , PS), systems based on helper viruses have already been described (Parks.
  • helper-dependent adenovirus vector system re oval of helper virus by cre-mediated excision of the viral packaging signal, Proc. Natl. Acad. Be. USA 93: 13565-13570) and are currently being further developed worldwide. Due to the size of the constructs, which places special demands on the cloning strategies, the cloning of minimal viruses is currently a complex and time-consuming process.
  • the object of the invention is to provide a cloning system with which the production of adenoviral minimal viruses is simplified and to insert new components which allow the use of these vectors in vitro and in vivo.
  • adenoviral minimal viruses The cloning of the adenoviral minimal viruses was simplified here by using cosmid cloning (see CI4.). The mechanism for virus formation created here does not restrict the choice of transgenes (see CI3.). In addition, a novel titer system was constructed, which allows the in vivo use of the adenoviral minimal viruses allowed (see CI5.2.). In addition, pMVX contains a fragment of chromosomal human DNA as a viral backbone to improve stability (see C.2.6.1.) And flexibility in the choice of the Fre d DNA to be incorporated (see C.2.6.2.). Both vectors are compatible with all helper systems described so far for the propagation and packaging of minimal viruses.
  • the bacterial backbone of the plasmid comes from pBluescript and contains, in addition to the origin of replication, a beta-lactamase gene for mediating ampicillin resistance.
  • the beta lacta asegen enables the use of conventional selection methods for the multiplication of pMV and its derivatives and their cloning.
  • the origin of replication of pBluescript is subject to a multi-copy control in E. coli and thereby enables the preparative multiplication of the minimal virus vector with high yield in suitable E. coli strains.
  • the plas id contains the 5 ' ITR with the packaging signal (the first 514 base pairs (bp) of the adenovirus genome) and the 3 ' ITR (the last 162 bp of the adenovirus genome). These are the minimally required signal sequences for the recognition of the minimal virus genome by the replication machinery of the adenoviral helper virus as well as the packaging of the replicated minimal virus genomes in viral capsids.
  • Minimal virus production is a mandatory basis for the production of minimal viruses and is therefore also described by others.
  • the release of the ITRs enables them to be effectively recognized by the replication apparatus.
  • the 18 bp recognition sequence conveys the highest specificity for the release of the ends and at the same time maximum flexibility in the choice of the transgenic sequences.
  • An 18 bp sequence statistically occurs only once in 6.8 x 10 11 base pairs, the human genome as the source of potential transgenes contains only approx. 3 x 10 9 base pairs. It can therefore be practically ruled out that a recognition sequence occurs in a desired transgene which would interfere with the release mechanism.
  • the phage La bda cos signal to facilitate cloning
  • Minimal virus genomes have the greatest stability if their genome size is as close as possible to that of wild-type adenovirus (approx. 36 kilobase pairs (Kb)). In this size range, the classic cloning processes are very inefficient and prone to errors.
  • the construct receives the properties of a cosmid vector through the cos signal.
  • cosmid cloning techniques enables a highly efficient and size-selected construction of recombinant minimal virus genomes.
  • the size selection results from the packaging capacity of the lambda phage, which, after subtracting the bacterial part in the plasmid, corresponds exactly to the minimum virus size range of approx. 25-38 Kb that is considered stable (Mitani, K. et al., 1995, Rescue, propagation , and partial purification of a helper virus-dependent adenovirus vector, Proc. Natl. Acad. Sci. USA 92, 3854-858). So far, the cosmid cloning technique has mainly been used for the production of gene banks. Your application to simplify minimal virus cloning is new.
  • Minimal viruses are not capable of independent replication, so titering cannot be carried out using the classic adenovirus titering method.
  • Minimal viruses that were generated from pMV and its derivatives can be titered by reporter genes, which enable direct visualization of infected cells via enzymatic color reactions or immunological detection methods. There is a choice between two reporter systems, one of which is new:
  • a constitutive beta-galactoidase expression cassette is inserted in pMV-BG. It consists of the beta-galactosidase gene under the control of the RSV-3 ' LTR (RSV promoter) and a downstream polyadenylation signal. Infected cells can be visualized by X-Gal staining.
  • the beta-galactosidase reporter system for visualizing viral infection and for titering viruses is a general procedure and has been used in gene transfer vectors various types, including adenoviral minimal viruses, already used.
  • Beta-galactosidase is a potent immunogen in vivo.
  • an alternative titer system is offered in pMV-PLAP, which is based on a reporter gene system, and which does not appear as an immunogen in vivo.
  • the regulated reporter gene cassette contains two components that ensure the lowest possible immunogenicity:
  • Reporter gene expression is controlled by the tet promoter, which is currently considered the most regulatable promoter in mammalian cells (Gossen, M. & Bujard, H., 1992, Tight control of gene expression in mam alian cells by tetracycline-responsive promoters, Proc. Natl. Acad. Sci. USA 89, 5547-551)
  • the promoter is only active in the presence of the tet transactivator. In mammalian cells that do not naturally express the transactivator, the promoter is largely silent.
  • PLAP placenta-like alkaline phosphatase
  • PLAP placenta-like alkaline phosphatase
  • a reporter gene a well-characterized human reporter gene with established detection methods (Kumar-Singh, R. and Chamberlain, JS, 1996, Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells, Hum. Mol. Genet. 5, 913-921.), which has no known antigenic properties in humans.
  • both the two components tet promoter and PLAP gene
  • the underlying principle of a double strategy for avoiding an immune response regulated promoter that is silent in the target organism and use of a reporter gene that connects the genome of the target organism originated
  • titering gene therapy vectors generally novel.
  • pMV contains a multiple cloning site with unique interfaces for several restriction enzymes to facilitate the insertion of therapeutic DNA fragments. Taking into account the adenoviral packaging capacity, fragment sizes up to approx. 30 Kb can be inserted. Vectors for minimal virus production with a multiple cloning site have not yet been described.
  • the minimal virus vector pMVX has all components of pMV except the multiple cloning site, in whose place a piece of non-coding DNA from the human genome is inserted. This gives the vector new properties. PMVX is also offered with two different reporter gene cassettes.
  • pMVX contains a 27.4 Kb human DNA fragment X-chromosomal origin, the sequence of which is completely known and contains no coding regions. Undesired effects due to gene expression from this component can therefore largely be excluded. Compared to pMV, the following additional properties result:
  • the chromosomal fragment forms the backbone of the minimal viruses that can be derived from pMVX.
  • Existing systems use e.g. DNA of phage La bda as the backbone.
  • the minimal viruses filled in with lambda DNA are, as known from congress reports, unstable, while the insertion of chromosomal fragments has a stabilizing effect.
  • vectors for minimal virus generation which exploit this systematically in that different transgene cassettes can be installed in a constant chromosomal context, have not been described to date.
  • cassettes in a size range of up to 18 Kb can be inserted in pMVX with a cloning step, and at the same time a minimal virus genome size can be maintained, the highest Stability of the derived minimal virus ensures.
  • This flexibility with regard to the size of the cassette to be inserted is novel for minimal virus cloning vectors. All enzymes provide ends that are either already smooth or can be filled in with Klenow polymerase. Cloning over smooth DNA ends is preferred in the cosmid cloning used here.
  • Embedding the therapeutic cassette in the chromosomal context leads to more effective gene transfer through various mechanisms. This is primarily due to the fact that the human genomic DNA used in the adenoviral minimal viruses stabilizes the vector after transfer to the target cell.
  • minimal viruses derived from pMV and pMVX are without restriction for the whole Spectrum of adenoviral gene transfer can be used. They meet the safety criteria for gene therapy vectors and are compatible with all currently described helper systems for minimal virus propagation.
  • cDNA constructs as therapeutic expression cassettes, which are usually controlled by viral promoters. These constructs can cause unnaturally high expression of the transgene, which goes beyond the therapeutic scope and can damage the cell.
  • First generation adenoviruses were used for the production of transgenic mice (T ⁇ ukui et al., 1996, Transgenesis by adenovirus-mediated gene transfer into mou ⁇ e zona-free egg ⁇ , Nature Biotechnology 14, 982-985).
  • the construction of the corresponding homologous recombination constructs with the vectors described here provides the possibility of inserting longer homologous regions due to the considerably increased absorption capacity of minimal viruses for foreign DNA, which should lead to an increased recombination frequency.
  • the generation of transgenic animals in species whose genetic manipulation via ES cell technology is currently not yet possible could be made possible by a viral strategy using minimal viruses.
  • the ITRs and the packaging signal of adenovirus type 5 (Ad5) and the constitutive beta-galactosidase expression cassette were first cloned into the plasmid pSL1190 sold by Pharmacia.
  • the 3 ' -ITR was amplified with the starter oligonucleotides AD5ITR1 (contains an extension of a BglII site) and AD5ITR3 (contains an extension of an EcoRI site) and wt-Ad5-DNA as a template by polymerase chain reaction (PCR).
  • the resulting 176 bp PCR product was digested with BglII and EcoRI and into the corresponding ones Restriction interfaces in the multiple cloning site of pSL1190 are inserted.
  • the 5 ' ITR with the adjacent packaging signal of Ad5 was amplified by PCR with the starter oligonucleotides AD5ITR1 (contains an extension of a terminal BglII interface) and AD5ITR2 (contains an extension of a terminal Asp718 interface) and wt-Ad5 DNA as a template.
  • the resulting 545 bp PCR product was digested with BglII and EcoRI and inserted into the corresponding restriction sites in the multiple cloning site of pSL1190.
  • Ad5ITRl 5'- CGGAGATCTCATCATCAATAATATACCTTATTTTGG -3 '
  • Ad5ITR2 5'- GTGACGGTACCAACTCTACTCGCTGGCACTCAAG -3 '
  • the constitutive beta-galactosidase cassette - consisting of the RSV promoter, the beta-galactosidase gene and the SV40 polyadenylation signal - was released from pRSVBGSV40 (cloned by Mr. Peter Lenderr) by double digestion with Nrul and Xbal as a 4230 bp fragment and inserted into the compatible Hpal and Avrll restriction interfaces in the multiple cloning site of pSLITRPS (cf. 1.2).
  • the 3 ' ITR was obtained by double digestion with EcoRI and Asp718
  • Double digestion of the starting plasmid pSLITRPSBG with P ⁇ tl and Bglll released a 4780 bp fragment, which contains the 5 ' - ITR, the packaging signal and a directly adjoining constitutive beta-galactosidase expression cassette (consisting of RSV promoter, beta-galacto ⁇ idae Gene and SV40 polyadenylation signal) contains. This was inserted into the multiple cloning site of pBSITR via the corresponding restriction interfaces.
  • pMV-BG.PL there are unique interfaces for Bglll and Spei to accompany the ITRs.
  • the plasmid was digested with BglII and Spei into a 2952 bp fragment (containing the bacterial backbone) and a 4965 bp fragment (the beta-galactosidase cassette as well as the ITRs and the PS) containing) split. The fragments were isolated separately and the 5 " overhanging ends of the 2952 bp fragment were filled in to blunt ends by Klenow polymerase.
  • the directional insertion of the meganuclease interfaces was carried out by a four-fragment ligation of these two fragments with two synthetic DNA compound pieces which contain the recognition sequence of the meganuclease each contain a smooth end and an overhanging end (matching the smooth ends of the 2952 fragment or the overhanging Bglll and Spel ends of the 4965-bp fragment).
  • These DNA connectors were also hybridized with the oligonucleotide IScel-Rev the oligonucleotide IScel-Bglll or IScel-Spel generated.
  • the CoS sequence motif was removed from the cosmid vector pWE15 by double digestion with Clal and AccI as a 2367 bp fragment and inserted into the unique Nhel site in the bacterial backbone of pMV-Bg.MH. Since the ends of the enzymes used are not compatible, all fragment ends were blunt-ended with Klenow polymerase and then ligated. This resulted in two products with different insertion orientations of the cos sequence. Since the sequence is independent of orientation, one of the two orientations was selected arbitrarily.
  • the 27.4 kB fragment was extracted from a David Bauer, AG Genome Analysis, IMB Jena, provided Cos id (cos3H10) released by digestion with NgoMI.
  • the human genomic sequence between the 5 " ITR / PS and the beta-galactosidase expression cassette was inserted by insertion into the Ago restriction restriction interface of pMV-BG.COS, which is compatible with NgoMI.
  • pMVI-Bg like pMV-Bg, but (1) additionally loxP
  • pMV like pMV-Bg, but without Beta-Galakto ⁇ ida ⁇ e-
  • pMVI like pMV-Bg, but (1) additionally loxP
  • pMVIX-Bg like pMV-Bg, but (1) additionally loxP
  • pMVX like pMV-Bg, but without beta-galactosidase
  • pMVIX like pMV-Bg, but (1) additionally loxP

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Abstract

L'invention concerne des vecteurs de clonage utilisés pour préparer des adénovirus de type virus minimaux, comprenant a) deux séquences de réplication terminales inversées de type adénovirus (ITR, inverted terminal repeats = répétitions terminales inversées), qui ab) sont flanquées de deux interfaces pour une endonucléase de restriction avec une longue séquence d'identification supérieure à 8 Bp, et ac) entourent ac) un signal de conditionnement de type adénovirus, ad) un site de clonage multiple pour insérer des fragments thérapeutiques d'ADN dans lesquels sont clonés éventuellement en outre de l'ADN d'animal mammifère chromosomique non codant, ainsi que ae) éventuellement un site d'identification pour une recombinase se trouvant entre une des séquences de réplication terminales inversées et le signal de conditionnement de type adénovirus, af) éventuellement une cassette de gène rapporteur; ainsi que b) un squelette bactérien de plasmide avec origine de réplication et gène de résistance bactérien, dans lequel ba) est cloné un signal de conditionnement d'un bactériophage.
PCT/DE1998/001940 1997-07-10 1998-07-06 Vecteurs de clonage pour preparer des adenovirus de type virus minimaux WO1999002647A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98944984A EP1003895A2 (fr) 1997-07-10 1998-07-06 Vecteurs de clonage pour preparer des adenovirus de type virus minimaux
JP2000502148A JP2001509375A (ja) 1997-07-10 1998-07-06 最小アデノウイルスベクターを作製するためのクローニングベクター

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DE19729571.1 1997-07-10
DE19729571 1997-07-10
DE19744768.6 1997-10-10
DE19744768A DE19744768C2 (de) 1997-07-10 1997-10-10 Klonierungsvektoren für die Herstellung von adenoviralen Minimalviren

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WO1999002647A2 true WO1999002647A2 (fr) 1999-01-21
WO1999002647A3 WO1999002647A3 (fr) 1999-04-15

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Cited By (8)

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WO2000049166A2 (fr) * 1999-02-18 2000-08-24 Merck & Co., Inc. Systeme de production de vecteurs d'adenovirus dependant d'auxiliaires base sur l'utilisation d'endonucleases
EP1126031A1 (fr) * 2000-02-16 2001-08-22 DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung Vecteur viral non humain, dependent des virus auxiliaires
WO2002070720A1 (fr) * 2001-03-02 2002-09-12 Riken Vecteurs de clonage et methode de clonage moleculaire
US6573092B1 (en) 2000-10-10 2003-06-03 Genvec, Inc. Method of preparing a eukaryotic viral vector
EP1320621A2 (fr) * 2000-09-15 2003-06-25 Merck & Co., Inc. Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
EP1359217A1 (fr) * 2002-04-29 2003-11-05 The Trustees of The University of Pennsylvania Méthode pour l'amplification et production des virus intégrés dans l'ADN cellulaire de tissu
US7135187B2 (en) 1993-06-24 2006-11-14 Advec, Inc. System for production of helper dependent adenovirus vectors based on use of endonucleases
JP2011115167A (ja) * 1999-02-22 2011-06-16 Georgetown Univ 全身性遺伝子送達のための抗体フラグメント標的化イムノリポソーム

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WO1994023582A1 (fr) * 1993-04-08 1994-10-27 Genetic Therapy, Inc. Vecteurs adenoviraux renfermant l'adn codant une proteine surfactant des poumons

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7135187B2 (en) 1993-06-24 2006-11-14 Advec, Inc. System for production of helper dependent adenovirus vectors based on use of endonucleases
WO2000049166A3 (fr) * 1999-02-18 2001-04-12 Merck & Co Inc Systeme de production de vecteurs d'adenovirus dependant d'auxiliaires base sur l'utilisation d'endonucleases
WO2000049166A2 (fr) * 1999-02-18 2000-08-24 Merck & Co., Inc. Systeme de production de vecteurs d'adenovirus dependant d'auxiliaires base sur l'utilisation d'endonucleases
JP2011115167A (ja) * 1999-02-22 2011-06-16 Georgetown Univ 全身性遺伝子送達のための抗体フラグメント標的化イムノリポソーム
US8859274B2 (en) * 1999-02-22 2014-10-14 Synergene Therapeutics, Inc. Antibody fragment-targeted immunoliposomes for systemic gene delivery
EP1126031A1 (fr) * 2000-02-16 2001-08-22 DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung Vecteur viral non humain, dependent des virus auxiliaires
EP1320621A2 (fr) * 2000-09-15 2003-06-25 Merck & Co., Inc. Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
EP1320621A4 (fr) * 2000-09-15 2005-11-23 Merck & Co Inc Vaccins adenoviraux de premiere generation evolues, exprimant les proteines gag, pol et nef du vih-1 a optimisation des codons et leurs modifications
US6573092B1 (en) 2000-10-10 2003-06-03 Genvec, Inc. Method of preparing a eukaryotic viral vector
US6908762B2 (en) 2000-10-10 2005-06-21 Genvec, Inc. Method of preparing a eukaryotic viral vector
WO2002070720A1 (fr) * 2001-03-02 2002-09-12 Riken Vecteurs de clonage et methode de clonage moleculaire
US7235393B2 (en) 2002-04-29 2007-06-26 The Trustees Of The University Of Pennsylvania Method for direct rescue and amplification of integrated viruses from cellular DNA of tissues
EP1359217A1 (fr) * 2002-04-29 2003-11-05 The Trustees of The University of Pennsylvania Méthode pour l'amplification et production des virus intégrés dans l'ADN cellulaire de tissu

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EP1003895A2 (fr) 2000-05-31
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