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WO1999041400A1 - Procedes de fabrication de vecteurs pseudo-adenoviraux - Google Patents

Procedes de fabrication de vecteurs pseudo-adenoviraux Download PDF

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Publication number
WO1999041400A1
WO1999041400A1 PCT/US1999/003483 US9903483W WO9941400A1 WO 1999041400 A1 WO1999041400 A1 WO 1999041400A1 US 9903483 W US9903483 W US 9903483W WO 9941400 A1 WO9941400 A1 WO 9941400A1
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Prior art keywords
adenovirus
genome
helper
packaging signal
packaging
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PCT/US1999/003483
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English (en)
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Samuel C. Wadsworth
Helen Romanczuk
Richard J. Gregory
Donna Armentano
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Genzyme Corporation
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Application filed by Genzyme Corporation filed Critical Genzyme Corporation
Priority to JP2000531581A priority Critical patent/JP2002538758A/ja
Priority to EP99908233A priority patent/EP1054989A1/fr
Priority to AU27718/99A priority patent/AU752284B2/en
Priority to CA002318737A priority patent/CA2318737A1/fr
Publication of WO1999041400A1 publication Critical patent/WO1999041400A1/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
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the invention is directed to novel helper adenoviruses which facilitate the production of pseudoadenoviral vectors (PAV) but which cannot be packaged into viral particles.
  • the invention is further directed to novel PAV producer cell lines expressing DNA binding and/or repressor proteins that prevent the packaging of the helper viruses.
  • the invention is also directed to methods for the production of PAV in such cell lines with minimal contamination from helper viruses.
  • Pseudoadenoviral vectors are adenoviral vectors derived from the genome of an adenovirus which contain the minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (see, e ⁇ , allowed U.S. Application Serial No. 08/895,194) incorporated herein by reference).
  • Such PAVs are advantageous because the transgene carrying capacity of the vector is optimized (up to 36Kb in size), while the potential for host immune reaction to viral proteins or for the generation of replication-competent viruses is reduced.
  • PAVs contain the adenoviral 5 1 and 3' inverted terminal repeat (ITR) nucleotide sequences containing origins of replication, the 5' cis-acting packaging signal of the viral genome, and can accomodate one or more transgenes with operably linked expression elements. These minimal viral nucleotide sequences retained in PAVs are required in cis for the replication and packaging of the PAV genome into viral particles. In addition, the production of PAVs requires the provision of a helper adenovirus to supply the viral proteins required for replication of the PAV genome and assembly of the viral particles.
  • ITR inverted terminal repeat
  • Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies in classical genetics and molecular biology (Horwitz, M.S., "Adenoviruses,” in Virology. 3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hitt, M.M. et al., "Adenovirus Vectors," in The Development of Human Gene Therapy. Friedman, T.
  • the viral genes are classified into early (known as E1-E4) and late (known as L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.
  • the human adenoviruses are classified into numerous serotypes (approximately 47, numbered accordingly and organized into 6 subgroups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, DNA base and protein amino acid compositions and homologies, and antigenic relationships.
  • Recombinant adenoviruses have several advantages for use as gene transfer vectors, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (Hitt, et al. supra): Berkner, K.L., Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
  • the cloning capacity of an adenovirus vector to date has been proportional to the size of the adenovirus genome present in the vector.
  • a cloning capacity of about 8 kb results from the deletion of regions of the virus genome which are dispensable for virus growth, e ⁇ , E3, and the deletion of a genomic region such as El whose function may be restored in trans from a complementing cell line such as 293 cells (Graham, F.L., J. Gen. Virol. 36:59-72, 1977).
  • Such El-deleted vectors are rendered replication- defective, a desirable attribute for a gene transfer vector.
  • the upper limit of vector DNA capacity for optimal carrying capacity is about 105%- 108% of the length of the wild-type genome.
  • adenovirus genomic modifications are possible in vector design using cell lines which supply other viral gene products in trans, e.g.. complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995).
  • transgenes foreign nucleic acids
  • first and second-generation adenoviral vectors illustrating the heterogeneity of adenoviral vector transduction.
  • transgenes include p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin (Descamps et al., Human Gene Therapy 5:979-985, 1994; ornithine transcarbamylase (Stratford-Perricaudet et al., Human Gene Therapy 1:241-256, 1990; We et al., J. Biol. Chem. 271;3639-3646, 1996;); adenosine deaminase
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Adenovirus vectors engineered to carry the CFTR gene have been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) and studies have shown the ability of these vectors to deliver CFTR to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:75-83, 1994), and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics 8:42-51, 1994).
  • first and second generation adenovirus vectors in gene transfer studies to date indicates that persistence of transgene expression in target cells and tissues is often transient. This is at least partly due to the generation of a cellular immune response to viral proteins which are expressed even from a replication-defective vector, triggering a pathological inflammatory response which may destroy or adversely affect the adenovirus-infected cells (Yang et al., J. Virol. 69:2004-2015, 1995; Yang et al., Proc. Natl. Acad. Sci. USA 91:4407-4411, 1994; Zsengeller et al., Hum Gene Ther. 6:457-467, 1995; Worgall et al, Hum. Gene Ther. 8:37-44, 1997; Kaplan et al., Hum. Gene Ther.
  • Modifications to the adenovirus genome in the recombinant vector can decrease the host immune response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al., J. Virol. 70:8944-8960, 1996; Gorziglia et al, J. Virol. 70:4173-4178; Kochanek et al, Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
  • the adenovirus E3 gpl9K protein can complex with MHC Class I antigens and retain them in the endoplasmic reticulum, which prevents cell surface presentation and killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al., Trends Microbiol. 437-443, 1994), suggesting that the presence of its encoding gene in a recombinant adenoviral vector may be beneficial.
  • CTLs cytotoxic T-lymphocytes
  • adenoviral vector-delivered transgenes may also be due to limitations imposed by the choice of promoter and/or transgene contained in the transcription unit (Guo et al, Gene Therapy 3:802-801, 1996; Tripathy et al., Nature Med. 2:545-550, 1996. Further optimization of minimal adenoviral vectors for persistent transgene expression in target cells involves the synergistic choice of expression control elements and vector genome design such that expression is maximized and host immune response is limited (WO98/46780 Scaria et al., J.Virol. 72:7302-7309, 1998).
  • PAV requires the presence of adenovirus proteins in trans which facilitate the replication and packaging of a PAV genome into viral vector particles.
  • adenovirus proteins are provided by infecting a producer cell with a helper adenovirus containing the genes encoding such proteins.
  • helper viruses are potential sources of contamination of a PAV stock during purification if they are able to replicate and be packaged into viral particles. It is advantageous, therefore, to increase the purity of a PAV stock by reducing or eliminating any production of helper viruses which can contaminate the preparation.
  • Several strategies to reduce the production of helper viruses in the preparation of a PAV stock are disclosed in allowed U.S. Patent Application Serial No. 08/895,194 and U.S. Patent No. 5,670,488, issued September 23, 1997, incorporated -6-
  • the helper virus can contain mutations in the packaging sequence of its genome which prevent packaging, or may contain an oversized adenoviral genome which cannot be packaged due to size constraints of the virion.
  • Other strategies include the design of a helper virus with a packaging signal flanked by the excision target site of a recombinase, such as the cre-lox system (Parks et al, Proc. Natl. Acad. Sci.USA 93:13565-13570, 1996; Hardy et al., J.Virol. 71:1842- 1849, 1997).
  • adenovirus packaging signal Detailed analysis of the structure of the adenovirus packaging signal has revealed that it is organized into a minimum of seven functional elements, identified as A repeats (Schmid et al, J. Virol. 71 :3375-3384, 1997).
  • a PAV production system which comprises helper adenoviruses and producer cell lines optimized for PAV production is provided such that the packaging signal region is available for the production of a helper virus stock but is disabled during the production of a PAV vector stock.
  • the present invention provides for an increased preferential packaging of PAVs in the production of purified vector stocks to further the development and widespread use of these vectors for gene transfer.
  • the invention is directed to novel helper adenoviruses which facilitate the production and packaging of pseudoadenoviral vectors (PAV) by providing for the production of essential viral proteins in trans production and packaging required for PAV the helper viruses of the inventors are packaging defective due to the inclusion in the packaging signal region of their genomes of binding sequences for DNA binding and/or repressor proteins that prevent access by packaging proteins to this signal.
  • the invention is also directed to to PAV producer cell lines expressing such nucleic acids encoding DNA binding and/or repressor proteins.
  • the invention is further directed to methods for the production of PAVs with minimal contamination from helper viruses, using the helper viruses and producer cell lines of the invention.
  • Figure 1 shows a schematic diagram of strategies for the production of packaging- defective helper adenoviruses.
  • Figure 2 shows a schematic diagram of the FLP recombinase/FRT helper system for excision of packaging sequences from helper adenoviruses.
  • Figure 3a shows a schematic diagram of a helper adenovirus containing a packaging signal flanked by lambda operator binding sequences;
  • Figure 3b shows the structure of the junction of the packaging signal and operator sequences.
  • Figure 4a shows a schematic diagram of a helper adenovirus containing a packaging signal flanked by lambda operator binding sequences
  • Figure 4b shows the structure of the junction of the packaging signal and operator sequences.
  • Figure 5 shows a schematic diagram of a helper adenovirus containing a packaging signal flanked by lambda operator binding sequences.
  • Figure 6 shows a schematic diagram of a helper adenovirus containing a packaging signal flanked by lambda operator binding sequences.
  • Figure 7 shows a schematic diagram of a series of helper adenoviruses which contain a packaging signal flanked by FRT binding sequences.
  • Figure 8 shows a schematic diagram of Ad2HelpFRT containing a packaging signal flanked by FRT binding sequences.
  • Figure 9 shows a schematic diagram of Ad2HelpFRTluc containing a packaging signal and luciferase marker gene flanked by FRT binding sequences.
  • Figure 10 shows a schematic diagram of helper adenovirus TBTP.
  • Figure 11 is a schematic diagram of a helper adenovirus with internal ITR sequences facilitating virus replication.
  • Figure 12 shows a schematic diagram of plasmid pTRE/FLPe6.
  • Figure 13a shows a schematic diagram of a tetracycline induction system for FLPe ⁇ expression;
  • Figure 13b shows tetracycline induction system for FLPe ⁇ expression in 293- tet-OFF cells.
  • Figure 14 shows a schematic diagram of plasmid pTRE/FLPe6.
  • Figure 15 shows a schematic diagram of plasmid pCEP/FLPe6.
  • Figure 16 shows a schematic diagram of plasmid pOG-FLPe6.
  • the invention is directed to novel helper adenoviruses which facilitate the production and packaging of pseudoadenoviral vectors (PAV) by providing for the production of essential viral proteins in trans required for PAV production and packaging.
  • the helper viruses of the invention are packaging defective due to the inclusion in the packaging signal region of their genome of binding sequences for DNA binding and/or repressor proteins that either prevent access by packaging proteins to this signal region or which facilitate enzymatic excision of the packaging signal.
  • the invention is also directed to to PAV producer cell lines expressing nucleic acids encoding such DNA binding and/or repressor proteins.
  • the invention is further directed to methods for the production of PAV with minimal contamination from helper viruses, using the helper viruses and producer cell lines of the invention.
  • a helper virus of the invention is defined as an adenovirus which is able to supply the viral proteins required in trans for the production of PAV or other minimal adenoviral vectors.
  • the helper virus genome is disabled for packaging, thereby allowing for preferential packaging of the PAV genomes into viral particles.
  • the helper virus genome comprises at least those genes and/or regions of the adenovirus genome that are required to produce the viral proteins required in trans for the production of PAV.
  • the adenovirus proteins supplied by the helper virus include inter alia the regulatory proteins from the adenovirus early (E) genomic regions, the capsid proteins encoded by the viral late (L) genomic regions, and other structural and non- structural proteins.
  • helper virus genome facilitates the replication of the PAV genome during the production of vector stock.
  • the adenovirus genes required in trans are not limited by virus serotype, and the helper viruses of the invention can contain adenovirus genes from more than one serotype.
  • Structural proteins which are supplied by the helper virus, can therefore be chosen so that the capsid proteins are derived from a desired serotype or serotypes and optimized for a particular use.
  • helper virus genome is desirably modified according to the present invention such that packaging of helper virus particles is impaired or eliminated. Such disability -9-
  • a helper virus of the invention is rendered packaging-defective by incorporation into its genome of a reduced length packaging signal or the insertion therein of heterologous nucleotide sequences (defined as binding sequences) into in or near the packaging signal region of the helper virus genome.
  • binding sequences are capable of binding to specific DNA binding and/or repressor proteins, thereby either blocking the utilization of the cis-acting packaging signal (packaging-signal masked) or causing excision of the signal from the helper virus genome (packaging-signal deleted) ( Figure 1).
  • a binding protein is defined herein as any protein or peptide (1) which is capable of binding to the binding sequences inserted into or near the packaging signal region of a helper virus genome so as to prevent utilization of the signal and repress packaging or (2) which is capable of binding to and induce cleavage at specific target sites.
  • Binding sequences are defined as nucleotide sequences inserted in proximity to, adjacent to or into the packaging signal region of the helper virus genome which are capable of binding to specific DNA binding and/or repressor proteins.
  • a binding sequence of the invention is capable of avidly binding one or more DNA binding and/or repressor proteins such that the packaging signal of a helper virus genome cannot be accessed by the viral packaging proteins or becomes excised from the genome.
  • the binding sequences interact with and bind DNA binding and/or repressor proteins.
  • the binding sequences which are incorporated into or near or near the packaging signal region of the helper virus genome can be of various lengths, e.g.. from about 8-30 nucleotides, but can also be tandem arrays of such sequences.
  • Preferred specific binding sequences of the invention include, but are not limited to, those derived from the bacteriophage lambda operator (Ptashne, M. A Genetic Switch. Cell Press and Blackwell Scientific Publications, 1986) and the papillomavirus E2 binding sequence (McBride et al., J.Biol. Chem. 266:18411-18414, 1991; Androphy et al., Nature 235:70-73, 1987). Most preferably, the packaging signal region in a helper -10-
  • flanked virus is flanked by the recognition site for a FLP recombinase, an enzyme which recognizes the FLP recombination target (FRT) and can catalyze site-specific excision of flanked nucleotide sequences (Senecoff et al., Proc. Natl. Acad.Sci. 82:7270-7274, 1985; O'Gorman et al., Science 251:1351-1355, 1991).
  • FRT FLP recombination target
  • the flanked packaging signal is excised from the helper virus genome, thereby preventing the packaging of the helper virus genome and the production of helper virus particles ( Figure 2).
  • the FLP recombinase is used in a producer cell of the invention for enzymatic cleavage of the packaging signal in a helper virus because it exhibits increased thermostability at 37°C (Buchholz et al., NAR 24:4256-4262, 1996; Buchholz et al.,
  • the invention contemplates the use of an FLP recombinase which is optimized for particular uses as needed in the form of monomers, dimers, tetramers or other multimeric forms.
  • Specific binding sequences which can be inserted in to an adenovirus genome at a site located in or near the adenovirus packaging signal region to accomplish the goals of the invention include the following:
  • O L l TATCACCGCCAGTGGTA (SEQ ID NO: 1) ATAGTGGCGGTCACCAT
  • the binding sequence is O L l:
  • Specific binding sequences for use by the papilloma virus E2 proteins include:
  • the FRT binding sequences can be derived from plasmid pOG45 (Stratagene, La Jolla,CA) or can be synthesized using standard techniques for oligonucleotide synthesis. Truncated FRT sequences which also facilitate cleavage of an adjacent packaging signal in a helper virus are also within the scope of the invention. The use of any binding sequences which facilitate FLP-mediated excision of a packaging signal from a helper adenovirus is within the scope of the invention in the construction of packaging-defective helper adenoviruses.
  • the packaging signal region which defines a minimum of seven AT-rich elements, denoted AI-AVII.
  • These seven AT-rich elements are located in the adenovirus genome from nucleotides 194-380 (referencing adenovirus serotype 5, Schmid et al., J. Virol. 71 :3375-3384, 1997).
  • one or more binding sequences can be inserted into one or more sites flanking the A elements, or can be inserted into or between the A repeats.
  • the redesign of the adenovirus packaging signal region according to the present invention also contemplates the deletion or multiplication of one or more A repeat elements.
  • binding sequences facilitate the excision of a packaging signal
  • they may be inserted into the adenovirus genome in order to flank a desired packaging signal region at the 5' and 3' ends thereof, such that the entire signal region is excised by a site-specific recombinase.
  • the helper virus genome is modified by the deletion of packaging elements AI-AIV, retaining only the packaging elements AV, AVI and AVII.
  • a binding sequence comprising a 17 bp bacteriophage lambda operator sequence (preferably, O L l) is inserted into the helper genome upstream and downstream from elements AV and AVII (sites #1 and #2), adjacent to nucleotides 334 and 385 of the adenovirus genome ( Figure 3:Ad(AV-VI- AVII).
  • the A repeat elements AV, AVI and AVII can be repeated as a motif, e.g., (AV-AVI-AVII) 2 and flanked by inserted lambda operator sequences as shown in Figure 4:Ad(AV-AVI-AVII) 2 .
  • Helper vectors Ad(AV-AVI-AVII) and Ad(AV- AVI-AVII) 2 can be used directly as helpers [packaging impaired, in the case of Ad(AV-
  • packaging signal region of a helper virus genome is modified by the deletion of packaging elements AI-AII-AIII-AIV, retaining only the packaging elements AV, AVI and AVII.
  • a binding sequence comprising a 17 -13-
  • bp operator sequence (preferably, O L l) from bacteriophage lambda is inserted into the helper virus genome between elements AV and AVI (site #3), as well as upstream of AV and downstream from AVII (sites #1 and #2) ( Figure 5).
  • the packaging signal region of a helper virus genome is modified by the deletion of packaging elements AVI and AVII.
  • a binding sequence comprising a 17 bp operator sequence from bacteriophage lambda (preferably, O L l) is inserted into the helper virus genome between All and AIII (site #5), as well as upstream from Al and downstream from AV (sites #4 and #6) ( Figure 6).
  • Preferred sites within the packaging signal region of the helper virus genome for insertion of any binding sequences include, but are not limited to:
  • binding sequences that bind, papilloma virus E2 protein are inserted into the helper virus genome to flank all or part of an packaging signal region in order to bind the E2 protein. Insertion sites set forth in Table 1 are preferred.
  • binding sequences comprise a lambda operator sequence used with the lambda repressor to prevent the packaging of the helper virus during the production of
  • binding sequences comprise a FRT binding sequence
  • specific designs of the packaging signal region of a helper virus genome include, but are not limited to ( Figure 7): FRT(V-VI-VII)FRT FRT(V-VI-VII) 2 FRT
  • a preferred embodiment of a helper adenovirus which comprises the packaging signal regions FRT(VI) 12 FRT or FRT(V-VI-VII) 2 FRT includes Ad2HelpFRT ( Figure 8).
  • helper viruses which are designed to reduce or prevent recombination between nucleotide sequences in a helper virus packaging signal region and the PAN or between a packaging signal region or other regions in a helper virus genome and the adenovirus genome sequences in a packaging cell line, e ⁇ , El sequences in 293 cells (nucleotides 1-4344 of adenovirus serotype 5), so as to prevent the generation of viable helper adenoviruses upon recombination.
  • helper virus genome comprises binding sequences that flank the packaging signal region (e.g.. lox sites which can be cleaved by the ere enzyme provided by a packaging cell line, Parks et al., Proc. ⁇ atl.Acad.Sci.USA 93:13565-13570, 1996, or contain the FRT target sites for the FLP recombinase), recombination between homologous nucleotide sequences in the helper virus genome and a producer cell could lead to the deletion of the flanking target sequences, e.g, the lox sites or the FRT sites.
  • flank the packaging signal region e.g. lox sites which can be cleaved by the ere enzyme provided by a packaging cell line, Parks et al., Proc. ⁇ atl.Acad.Sci.USA 93:13565-13570, 1996, or contain the FRT target sites for the FLP recombinase
  • helper virus genome contains one or more AVI packaging signal sequences (reference for signal elements, Schmid et al, J. Virol. 71:3375-3384, 1997) which only minimally overlap with any nucleotide sequences in a PAV vector (e.g.. nucleotides 1-356, containing only packaging signal elements AI-AV; disclosed in allowed U.S. Patent Application Serial No. 08/895,194 and U.S. Patent No. 5,670,488).
  • the possibility of recombination between the PAV and the helper virus genome is significantly reduced.
  • the signal region comprises a (12 x AVI repeats) nucleotide sequence, or contains tandem repeats of this -16-
  • helper virus genome contains no overlapping nucleotide sequences with a PAV genome, which utilizes a packaging signal containing nucleotides sequences from the A repeats I-V.
  • a helper packaging signal region can be flanked by binding sequences that facilitate excision of the packaging signal by a recombinase (e.g.. lox, FRT) or by binding sequences that facilitate binding to the packaging signal by a repressor protein (e.g.. lambda repressor) in accordance with the invention.
  • a marker gene also may be inserted into a helper virus packaging signal region in order to act as a marker for the excision of the packaging signal by loss of the characteristic signal encoded by the marker gene, e.g., luciferase (assay kits available from Promega, Madison, Wl).
  • luciferase assay kits available from Promega, Madison, Wl.
  • a preferred genome of a marker helper adenovirus is
  • Ad2HelpFRTluc ( Figure 9) which is constructed such that, upon excision of the FRT- flanked packaging signal by an FLP recombinase, the signal from a luciferase protein can no longer be detected.
  • recombination with homologous nucleotide sequences in a producer cell line would also result in loss of the marker signal, although the packaging signal will not be deleted. Therefore, a marker helper adenovirus is therefore most optimally used in a producer cell line which does not contain nucleotide sequences which may generate recombination with the 5' region in a helper virus and confound interpretation of a marker assay for excision of a packaging signal by an FLP recombinase.
  • a marker helper adenovirus may be used in a cell line, such as PER.C6 cells (containing adenovirus serotype 5 nucleotides 459-3510), whose genome comprises adenovirus sequences which only minimally overlap with any 5' nucleotide sequences in a helper virus, thereby reducing the likelihood of recombination (Fallaux et al, Hum.Gene Ther. 1:1909-1917, 1998).
  • PER.C6 cells are preferably used in the production methods of the invention in order to minimize recombination between producer cells and a helper adenovirus.
  • helper virus that reduces the possibility of recombination between the helper virus genome and the overlapping adenovirus nucleotide sequences in a complementing cell line (e.g.. El sequences in 293 cells) or in a PAV that can result in the production of viable helper viruses which contaminate a PAV preparation.
  • the genome of helper virus comprises a packaging -17-
  • the packaging signal of such helper virus comprises any combination of the A repeat elements (Schmid et al., J. Virol. 71 :3375-3384, 1997) sufficient to confer packaging capability, inserted into the genome in a reverse orientation.
  • the packaging signal region includes a (12 x AVI repeats) sequence in a reverse orientation.
  • a helper virus is provided which is inactivated upon the acquisition of homologous sequences from a recombination event with a complementing cell line, preferably an El -complementing cell line, such as 293 cells.
  • a complementing cell line preferably an El -complementing cell line, such as 293 cells.
  • this helper virus is engineered such that its size, upon a recombination event, renders the virus genome too large for packaging.
  • a preferred embodiment is the helper virus TBTP whose genome contains a FRT-flanked packaging signal ( Figure 10), and in which the E3 region of the adenovirus genome is deleted for 2.9 kb, but into which the 1.8 kb EGFP gene operably linked to a CMV promoter and a 5.0 kb fragment of the human alpha-antitrypsin gene are inserted.
  • the size of the helper virus is 36.9 kb (102% of wild-type), but upon recombination with adenovirus El sequences in 293 cells, the helper virus genome size becomes approximately 39.6 kb (110%), which is too large to be packaged.
  • the adenovirus genome is modified by the insertion thereto of binding sequences in proximity to or into the A repeats of the packaging signal region so as to prevent access to the cis-acting packaging signal upon the binding of a DNA binding and/or repressor protein to the specific binding sequences.
  • Standard techniqes of molecular biology such as restriction enzyme digestion and ligation, polymerase chain reaction and site-directed mutagenesis can be used to create a recombinant packaging signal region within a helper adenovirus genome.
  • Such a packaging signal region can then be inserted into the appropriate site in the 5' region of an adenovirus genome utilizing a plasmid comprising the signal.
  • a plasmid can be co-transfected into a cell line with DNA encoding the remainder of the adenovirus helper genome to be contained in the -18-
  • helper virus such that homologous recombination occurs, thereby generating a helper adenovirus with the desired recombinant packaging signal.
  • the helper viral genome can also be constructed in bacteria, thus simplifying the procedure (Chartier et al., J.Virol. 70:4805-4810, 1998).
  • the entire viral genome can be provided by transfection of a plasmid from which any non-viral (i.e., bacterial) sequences have been removed.
  • Other methods for the production of recombinant adenoviruses are known to those skilled in the art and can be used to produce the helper viruses of the invention.
  • the helper viruses of the invention can be derived from any wild-type, truncated or mutant adenovirus whose genome encodes the viral proteins required in trans to produce PAVs, and are not limited by serotype.
  • the helper viruses of the invention are also replication-defective, as an additional safety feature for the use in generating PAVs for use in gene transfer.
  • Replication-defective viruses can be created by, for example, deletion of the El region of the adenovirus genome.
  • Such helper viruses can be propagated in El -complementing cell lines such as the 293 cell line (Graham et al., J.Gen.Nirol.36:59-72, 1977).
  • a helper virus genome contains an internal ITR sequence located downstream from the blocked packaging signal region which allows for adequate replication and expression of the helper virus genome in the PAN producer cell line, thereby ensuring adequate provision of the viral proteins required in trans.
  • a repressor protein can occupy the binding sequences inserted into or near a helper virus packaging signal, this particular embodiment of the invention provides an exposed ITR which is available to the adenovirus D ⁇ A polymerase and terminal protein for replication of the helper virus genome ( Figure 11).
  • a method for the production of PAN vectors which uses a PAN genome and a helper adenovirus that contain packaging signal regions or ITR sequences from different adenovirus serotypes such that sequence -19-
  • the invention is further directed to PAV producer cell lines that produce D ⁇ A binding and/or repressor proteins which can bind to the binding sequences inserted into or near the packaging signal region of the helper virus genome.
  • the helper virus is disabled for packaging or deleted during the production of PAV stocks.
  • 293 cells comprising and express a nucleic acid encoding a D ⁇ A binding and/or repressor protein, thereby creating a PAV producer cell line of the invention.
  • Other cell lines can be used, including, but not limited to, VK2-20, as well as any cell lines designed to complement deletions of adenoviral genomic regions E2a (Zhou et al., J. Virol.
  • 293 cells comprise and express a nucleic acid encoding a D ⁇ A binding protein, preferably the FLP or FLPe recombinase, under the control of the tetracycline gene control system (Gossen and Bujard, Proc. ⁇ atl.
  • TetR tetracycline repressor
  • TetR/VP16 herpes virus VP16 transcriptional activation domain
  • TetR/VP16 and rTetR/vpl ⁇ controls the expression of genes linked to a minimal promoter cloned adjacent to tetracycline transcriptional regulatory elements (TRE), such as a gene for FLP recombinase stably integrated into 293 cells.
  • TRE tetracycline transcriptional regulatory elements
  • a 293-Tet-off cell line which further expresses a FLP recombinase can also be used to inducibly and preferentially excise a helper adenovirus packaging signal region which is flanked by FTR binding sequences or could also be used to excise any desired segment of an adenoviral genome which is flanked by the requisite FRT binding sequences.
  • a nucleic acid encoding the FLP recombinase is cloned into a pTRE plasmid (Clontech, Palo Alto, CA) ( Figure 12), such that the FLP recombinase is under the control of a minimal CMV promoter operably linked thereto and seven tet operator sites which are responsive to the TetR-VP16 fusion protein ( Figure 13a).
  • a pTRE plasmid (Clontech, Palo Alto, CA)
  • Modulation of expression of the FLP recombinase, and therefore of FLP-catalyzed excision of a helper adenovirus packaging signal in a dose-dependent manner can occur relative to the level of tetracycline provided to the producer cell.
  • a producer cell line of the invention is a 293 cell line comprising a nucleic acid encoding Tet-off fusion protein and the FLP recombinase under the control of the TRE operator sequences and the mimmal CMV promoter such that the cell line provides modulated production of the FLP recombinase when provided with a PAV genome and a helper virus of the invention containing a packaging signal flanked by FRT binding sequences, such that the helper virus is preferentially packaging-disabled during PAV vector production.
  • Producer cell lines which stably express a nucleic acid encoding the FLP recombinase can also be constructed using plasmids which contain the gene encoding FLP recombinase under the control of any suitable promoter, such as CMV or SV40.
  • plasmids which contain a FLP recombinase gene are pSVK/FLPe6 (containing FLP recombinase gene under the control of the SV40 promoter) ( Figure 14) and pCEP/FLP36, containing FLP recombinase gene operably linked to the CMV promoter ( Figure 15). Where it is desirable to provide the FLP recombinase gene to a cell -21-
  • plasmid which is maintained extrachromosomally, e.g., pCEP/FLPe6 ( Figure 15) can be used.
  • Nucleic acids encoding the DNA binding and/or repressor proteins are engineered into PAV producer cell lines by standard techniques of molecular biology, and can be stably or inducibly expressed.
  • Preferred DNA binding and/or repressor proteins to be used in the invention include, but are not limited to, the bacteriophage lambda repressor (wild-type and/or N-terminal fragment) Cro protein (Ptashne, M. A Genetic Switch. Cell Press and Blackwell Scientific Publications, 1986); the bovine papillomvirus E2 protein (McBride et al, J.Biol. Chem.
  • DNA binding and/or repressor proteins which are capable of binding to the relevant binding sequences in a helper virus genome can be used in the cell lines of the invention. Fusion proteins of such repressor proteins can also be constructed, for example, by creating a nucleic acid encoding E. coli ⁇ -galactosidase linked to the lambda repressor protein, thereby providing a substantial physical obstacle to packaging of the helper virus genome when such a fusion protein binds to the binding sequence inserted into or near a packaging signal in a helper virus genome.
  • Fusion proteins can also be generated by creating a hybrid nucleic acid encoding a DNA binding and/or repressor protein fused to an activator protein (see tet repressor/ ⁇ SV VP16, Gossen et al., Proc.Natl.Acad.Sci. USA 89:5547-5551, 1992).
  • the lambda repressor protein monomer is 236 amind acids in length (26KD); a repressor protein dimer binds to one 17 bp lambda operator sequence.
  • the 6 lambda operator sites are recognized by the lambda repressor protein dimer in order of their intrinsic affinities for a lambda repressor dimer, each with a central base pair, the axis of symmetry.
  • the N-terminal protein domain of the lambda repressor recognizes the operator and can be used for binding; in one embodiment of the invention, the C-terminal -22-
  • domain of the repressor can be replaced with, for example, a dimeric leucine zipper protein.
  • a nucleic acid encoding a DNA binding and/or repressor protein and the nucleotide sequences for any operably linked regulatory elements are introduced into a cell line by any method of nucleic acid transfer, including, but not limited to, transfection, electroporation, or viral-mediated transfer.
  • a plasmid comprising a nucleic acid encoding a DNA binding and/or repressor protein and nucleotide sequences corresponding to any regulatory elements can be transfected into a cell of interest. If the plasmid further contains a nucleic acid encoding a selectable marker, integration of the exogenous plasmid DNA can be detected using such marker.
  • a nucleic acid encoding neomycin resistance can be introduced in parallel with the nucleic acid encoding a repressor protein, and the cells which are stably transfected thereby can be selected by cultivation in the presence of G418.
  • a nucleic acid encoding such the repressor protein can be provided to a cell on an extrachromosomal plasmid which is maintained episomally (e.g.. EBNA-based system), such as pCEP-4 (Invitrogen, San Diego, CA).
  • any binding sequence-repressor protein pair in the design of the helper viruses and cell lines of the invention which is able to effectuate a binding interaction that prevents utilization of the packaging signal in a helper virus genome or which facilitates the excision of the signal, thereby preventing packaging of the helper virus.
  • helper adenoviruses and producer cell lines are useful in high-level production of PAV, allowing for preferential packaging of PAV genomes into gene transfer vectors relative to the helper viruses, thereby providing a means to produce helper-dependent PAVs with minimal contamination by helper viruses.
  • the invention is also directed to methods for the production of PAVs in high yield, using the helper viruses and producer cell lines of the invention.
  • PAV is preferentially produced, generating an enriched preparation.
  • the PAV genome which comprises the adenovirus 5' ITR and packaging signal and 3' ITR, and further comprises one or more transgenes up to 36 kb in size, operably linked -23-
  • PAVs are preferentially packaged because the PAV genome contains a wild-type packaging signal, in contrast to the helper virus in which the packaging signal has been disabled or deleted.
  • plasmid When the PAV genome is delivered to a producer cell on a plasmid, such plasmid can be introduced into a cell line of the invention by any method of nucleic acid transfer, including, but not limited to, transfection, lipofection and electroporation.
  • the cell line can be infected with an adenovirus helper which is available to provide the adenovirus proteins needed in trans to produce and package the PAV genome.
  • 2-20 ⁇ g of DNA which encodes a PAV genome is delivered to a cell by lipofection using a kit such as Profectin (Promega, Madison, WI), and the cells are infected with a helper virus of the invention using a multiplicity of infection (MOI) from 0.5 to 10.
  • MOI multiplicity of infection
  • a PAV producer cell line comprises an integrated PAV genome, as well as a nucleic acid encoding DNA binding and/or repressor protein, such that the PAV genome can be conditionally excised and the nucleic acid encoding the binding and or repressor protein can be conditionally expressed.
  • a PAV vector genome flanked by lox nucleotide sequences is stably integrated into a PAV producer cell line which is engineered to express a nucleic acid encoding the Cre recombinase (Sternberg et al., J.Mol. Biol. 150:467-486, 1981; Sauer et al., Meth. Enzymol.
  • Cre recombinase and the repressor protein can be inducibly produced using one or more inducible promoters susceptible to induction by such agents as tetracycline or ecdysone, among others.
  • This strategy for the production of PAV requires only infection of the producer cell by a helper virus and induction of the recombinase and repressor proteins to generate a preferentially packaged PAV stock.
  • the use of other site-specific recombinases is also within the scope of this embodiment of the invention, e ⁇ ., the FLP recombinase and its target sequence, FRT.
  • Purification of PAVs from a cell line of the invention can be performed by standard techniques of virus purification known to those skilled in the art.
  • viruses in cell lysates from producer cells can be purified on a standard CsCl gradient.
  • the PAV particles are of lower density relative to the helper viruses and will band at a higher position in the gradient, allowing for direct isolation and recovery.
  • viruses in cell lysates from producer cells can be purified on a standard CsCl gradient.
  • the PAV particles are of lower density relative to the helper viruses and will band at a higher position in the gradient, allowing for direct isolation and recovery.
  • PAV purification can be performed using chromatographic techniques, e.g.. as set forth in published PCT Application WO97/08298, incorporated herein by reference.
  • PAV yield is calculated by measuring the DNA and protein composition of the purified preparation. Maizel et al. (Virology 36:115-125, 1968) determined that an adenovirus virion comprises 13% DNA, with the remainder being protein.
  • Transgene activity of a PAV preparation is monitored by immunoflourescent techniques by infecting 293 cells with the PAV helper, then using an antibody against a PAV-encoded transgene expression product (protein) to determine infectious particles.
  • enzyme activity encoded by a transgene can be measured (e.g.. ⁇ - galactosidase, -galactosidase, ⁇ -antitrypsin), by, for example, an ELIS A assay.
  • the ability of PAV to enter cells is determined by measuring the amount of viral capsids that bind to the cells with anti-adenovirus antibodies. Demonstration of the entry of the PAV genome into a cell can be performed by fluorescent in situ hybridization (FISH).
  • helper virus production if any, can be scored, for example, by standard plaque assays on 293 cells.
  • the ratio of PAV to helper virus will be greater than 10,000 to 1.
  • Plasmid pAd/ITR (l-194)Mlu2 was digested with Spel and Mlu I. Oligonucleotides containing a 5' ⁇ 3' FRT site upstream from two copies of the AV-AVI- AVII packaging sequence were annealed together and ligated into the Spel/Mlu I site of pAD/TTR(l-194)Mlu 2. The resulting vector was designated pAD/FRT(AV-AVI-AVII) 2 .
  • Oligonucleotides 4758 (5' - CGC GTG AAG TTC CTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA) (SEQ ID NO: 12) and 4759 (5' - CGC GTG AAG TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC GGA ATA GGA ACT TCA) (SEQ ID NO: 13), containing a second FRT site oriented in the same direction as the first, were annealed together and ligated into the Mlu I site of pAD/FRT(AV-AVI-AVII) 2 and designated pAD/FRT(AV-AVI-AVII) 2 FRT ( Figure 7).
  • oligonucleotides 4760 (5' - CGC GTC GCG TAA TAT TTG TCT AGG GCC GCG GGG ACT TTG ACC GTT TAG AAG TTC CTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA) (SEQ ID NO: 14) and 4761 (5' - CGC GTG AAG TTC CTA TAC TTT CTA
  • pAD/FRT(AVfl 1 /luc/FRT using oligonucleotides containing twelve copies of AVI Oligonucleotides containing a 5'-*3' FRT site upstream from twelve head-to-tail copies of the AVI packaging sequence were annealed together and ligated into the Spe I / Mlu I site of pAD/ITR(l-194)Mlu2, generating pAD/FRT(AVI) 12 pGL3 control vector (Promega, Madison, WI) was digested with Mlu I and Bam HI and the 2427 bp fragment containing the luciferase cDNA under the control of the SV40 promoter/enhancer elements was isolated.
  • Oligonucleotides containing a second FRT site were annealed together and coligated with a 2427 bp luciferase fragment into the Mlu I site of ⁇ AD/FRT(AVI) 12.
  • the resulting vector containing an FRT flanked (AVI) 12 /luc cassette was designated pAD/FRT(AVI) 12 /luc/FRT ( Figure 9).
  • pAD/FRT(AVD 1 FRT using an oligonucleotide containing one copy of AVI Head-to-tail copies of the AVI packaging sequence were constructed by concatomerizing oligonucleotides 4755 (5' - TCG ACC GCG GGG ACT TTG ACC) (SEQ ID NO: 16) and 4754 (5' - TCG AGG TCA AAG TCC CCG CGG) (SEQ ID NO: 17) in the presence of T4 DNA ligase.
  • reaction was digested with Xho I to eliminate head-to-head and tail-to-tail ligation products, and cloned into the Xho I site of pSLl 180 (Amersham Pharmacia Biotech, Piscataway, NJ) generating pSL/(AVI) 12 FRT sites flanking the AVI repeats were inserted in two consecutive cloning steps.
  • pSL/(AVI) 12 was digested with Spe I and partially digested with Xho I.
  • Oligonucleotides HR100 (5' - CTA GTG AAG TTC CTA TTC CGA AGT TCC TAT
  • Helper adenoviruses containing the above-described designs of the 5' adenovirus packaging signal region are constructed in vitro using standard ligation techniques or by homologous recombination in vivo with any desired adenovirus.
  • EXAMPLE 2 Construction of helper adenoviruses containing lambda operator sites Helper vector Ad(AV- A VI- AVII).
  • Adenovirus nucleotides 1 through 194 were isolated by PCR and cloned into pAdvantage (Genzyme Gene Therapy, Framingham MA), in place of Ad nucleotides 1 through 357.
  • pAdvantage encodes the Ad2 genome ( ⁇ El, E3 ⁇ 2.9) in pBR322.
  • Sequences encoding a deleted packaging site (AV- AVI- AVII) (nucleotides 334-385) were cloned into an Spel / Mlul site within the vector and flanked -28-
  • Helper vector Ad(AV-AVI-AVII) 2 was built similarly to Ad(AV- AVI- AVII) ( Figure 4a; junction sequences are shown in Figure 4b). This helper vector incorporates 2 copies of the (AV- A VI- A VII) packaging repeat sequences, giving it a packaging efficiency equivalent to wild-type
  • EXAMPLE 3 Construction of TBTP helper adenovirus
  • the E3 gene of Ad2 (nts 27970 - 30937) is replaced with a 1.8kb EGFP expression cassette (CMV / EGFP / SvpA), at a genetically engineered RsrII site in the E3-deleted genome.
  • CMV / EGFP / SvpA 1.8kb EGFP expression cassette
  • EXAMPLE 4 Construction of producer cells expressing FLP recombinase
  • Plasmids pFLPe ⁇ Plasmids pFLPe ⁇ .
  • pOG-Flpe6 was received from Francis Stewart (EMBL, Heidelberg, Germany) ( Figure 16). This gene contains mutations of the Flp gene that make the encoded protein more stable at 37 °C. The gene, with upstream and downstream regulatory elements, is cloned into pOG44 (Stratagene, La Jolla, CA) behind the CMV promoter ( Figure 16).
  • pSVK/FLPe ⁇ The 2.0 kb Xbal / Sail fragment of pOG-Flpe6 was cloned directly into the Xbal / Sail site of the 3.9 kb pSVK3 (Pharmacia, Piscataway, NJ).
  • the plasmid pSVK FLPe6 has the Flpe ⁇ gene under the control of both eukaryotic (SV40e) and Prokaryotic (T7) promoters ( Figure 14).
  • SV40e eukaryotic
  • T7 Prokaryotic
  • pCEP/FLPe6 2.0 kb Xbal / Sail fragment of pOG-Fl ⁇ e6 was cloned into the Acc65I / Xhol site of the 10.4 kb pCEP-4 (Invitrogen, San Diego, CA ) using adapter -29-
  • the plasmid pCEP/FLPe6 has the Flpe ⁇ gene under the control of the CMV promoter.
  • the plasmid retains the EBNA-1 gene and EBV origin for extrachromosomal plasmid maintenance and the hygromycin gene for plasmid selection ( Figure 15).
  • pTRE/FLPe ⁇ The 2.0 kb Xbal / Sail fragment of pOG-Flpe ⁇ was cloned into Acc65I / Xhol site of the 3.1 kb pTRE plasmid (Clontech, Palo Alto, CA) using adapter linkers.
  • the Flpe ⁇ gene in pTRE/FLPe ⁇ is under the control of a minimal CMV promoter (hCMV*) and 7 operator sites (tet O, 1 through 7).
  • Producer Cells pCEP/Flpe ⁇ cells 293 and PER.C6 cells are transfected with 10-20 ⁇ g of pCEP/Flpe ⁇ DNA. Following hygromycin selection, cells expressing high levels of Flpe ⁇ are selected based on a functional assay for FLP activity, such as FRT-mediated excision of a target sequence. Cells with a stable extrachromosomal plasmid copy number are selected for PAV amplification. pSVK/Flpe ⁇ cells. Cells are transfected similarly as described above, with the addition of a plasmid bearing a neo resistance gene marker.
  • cells expressing high levels of Flpe ⁇ are selected based on a functional assay for FLP activity, such as FRT-mediated excision of a target sequence.
  • Cells with stable, integrated pSVK/Flpe ⁇ DNA are selected for PAV amplification.
  • pTRE/Flpe ⁇ cells Cells are transfected as described above, with the addition of the pTK/hygro plasmid (Clontech, Palo Alto, CA) in the transfection.
  • pTK/hygro plasmid Clontech, Palo Alto, CA
  • cells expressing low background and high tetracyline-induced levels levels of Flpe ⁇ are selected based on a functional assay for FLP activity, such as FRT-mediated excision of a target sequence.
  • Cells with stable, integrated, inducible pTRE/Flpe ⁇ are selected for PAV amplification.
  • PAV DNA excised from backbone DNA (4-20 g) is used to transfect semi- confluent PER.C6 or 293 cells.
  • the cells express a repressor protein, which, upon binding to cognate sequences in -30-
  • helper vector prevents its packaging. After overnight incubation, cells are infected at an MOI of 1 to 10 with an El -deleted helper adenovirus harboring the appropriate packaging impaired sequences. Following the observation of complete cytopathic effect, cells and lysates are collected and the lysate is used for serial propagayion and expansion of PAV. Following several amplifications, the cell lysate is CsCl banded for the isolation of PAV.

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Abstract

L'invention concerne des adénovirus auxiliaires qui facilitent la production de vecteurs pseudo-adénoviraux (PAV) mais qui ne peuvent pas être encapsidés dans des particules virales. L'invention concerne également de nouvelles lignées cellulaires de production de PAV qui expriment les protéines liantes ou les répresseurs des ADN empêchant l'encapsidation des adénovirus auxiliaires. En outre, l'invention concerne des procédés de fabrication de PAV dans ces lignées cellulaires avec un minimum de contamination due aux virus auxiliaires.
PCT/US1999/003483 1998-02-17 1999-02-17 Procedes de fabrication de vecteurs pseudo-adenoviraux WO1999041400A1 (fr)

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JP2000531581A JP2002538758A (ja) 1998-02-17 1999-02-17 偽アデノウイルスベクターの製造方法
EP99908233A EP1054989A1 (fr) 1998-02-17 1999-02-17 Procedes de fabrication de vecteurs pseudo-adenoviraux
AU27718/99A AU752284B2 (en) 1998-02-17 1999-02-17 Methods for pseudoadenoviral vector production
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US7135187B2 (en) 1993-06-24 2006-11-14 Advec, Inc. System for production of helper dependent adenovirus vectors based on use of endonucleases
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WO2001021824A1 (fr) * 1999-09-23 2001-03-29 Genzyme Corporation Vecteurs auxiliaires et lignees cellulaires de production de vecteurs pseudoadenoviraux
WO2001053504A1 (fr) * 2000-01-21 2001-07-26 University Of Michigan Medical School Vecteurs adenovirus deficients pour la replication
US6867022B1 (en) 2000-01-21 2005-03-15 Regents Of The University Of Michigan Replication deficient adenovirus vectors and methods of making and using them
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CA2318737A1 (fr) 1999-08-19
JP2002538758A (ja) 2002-11-19
EP1054989A1 (fr) 2000-11-29
AU2771899A (en) 1999-08-30
AU752284B2 (en) 2002-09-12

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