WO2001021824A1 - Vecteurs auxiliaires et lignees cellulaires de production de vecteurs pseudoadenoviraux - Google Patents

Vecteurs auxiliaires et lignees cellulaires de production de vecteurs pseudoadenoviraux Download PDF

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WO2001021824A1
WO2001021824A1 PCT/US2000/025131 US0025131W WO0121824A1 WO 2001021824 A1 WO2001021824 A1 WO 2001021824A1 US 0025131 W US0025131 W US 0025131W WO 0121824 A1 WO0121824 A1 WO 0121824A1
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sequences
helper
genome
vector
pav
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Helen Romanczuk
Samuel C. Wadsworth
Patricia Berthelette
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Genzyme Corporation
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Priority to AU75791/00A priority Critical patent/AU7579100A/en
Priority to CA002388365A priority patent/CA2388365A1/fr
Priority to US09/718,034 priority patent/US6541245B1/en
Publication of WO2001021824A1 publication Critical patent/WO2001021824A1/fr

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    • 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
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    • 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
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    • 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
    • C12N2710/10351—Methods of production or purification of viral material
    • C12N2710/10352—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
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    • 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
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    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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 present invention is directed to improved adenoviral helper vectors that facilitate the production of pseudoadenoviral vectors (PAV), wherein the helper vectors themselves cannot be packaged into viral particles efficiently. While the helper vectors of the present invention are engineered, such that during PAV manufacture they are packaging defective, they may provide replication functions and viral structural proteins in trans for PAV.
  • the helper vectors of the present invention comprise recombinase protein recognition sequences, wherein said recognition sequences are inserted into regions of the helper vector genome to allow for separation of the replication and packaging elements of the helper vector.
  • the invention is also directed to improved cell lines for the production of PAV which facilitate PAV stock production.
  • the improved producer cell lines are stably transfected with a novel PAV.
  • the combination of the novel helper vectors of the present invention, the novel PAVs and the improved cell lines facilitate PAV stock production and make the invention adaptable to large scale commercial production of PAV stocks.
  • Adenoviral vectors for use to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro study and/or production of the products of transgenes commonly are derived from adenoviruses by deletion of the early region 1 (El) genes (Berkner, K.L., Curr. Top. Micro. Immunol. 158:39-66. 1992). Deletion of El genes renders such adenoviral vectors replication defective and significantly reduces expression of the remaining viral genes present within the vector.
  • El early region 1
  • adenoviral vectors can be deleterious to the transfected cell for one or more of the following reasons: (1) stimulation of a cellular immune response directed against expressed viral proteins, (2) cytotoxicity of expressed viral proteins, and (3) replication of the vector genome leading to cell death.
  • PAVs pseudoadenoviral vectors
  • PAVs are adenoviral vectors derived from the genome of an adenovirus that contain minimal exacting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes
  • Such PAVs which can accommodate up to about 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication-competent viruses is reduced.
  • PAV vectors contain the 5' inverted terminal repeat (ITR) and the 3' ITR nucleotide sequences that contain the origin of replication, and the cz ' _. -acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements, e.g. promoters, enhancers, etc.
  • Adenoviral vectors, such as PAVs have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for delivery of nucleic acids to recipient cells.
  • 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 (Hurwitz, M.S., Adenoviruses Virology, 3rd edition, Fields et al., eds., Raven Press, New York, 1996; Hitt, M.M. et al., Adenovirus Vectors. The Development of Human Gene Therapy, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York, 1999).
  • the viral genes are classified into early (designated E1-E4) and late (designated L1-L5) transcriptional units, referring to the generation of two temporal classes of viral proteins.
  • the demarcation of these events is viral DNA replication.
  • the human adenoviruses are divided into numerous serotypes (approximately 47, numbered accordingly and classified into 6 groups: A, B, C, D, E and F), based upon properties including hemaglutination of red blood cells, oncogenicity, DNA and protein amino acid compositions and homologies, and antigenic relationships.
  • Recombinant adenoviral vectors have several advantages for use as gene delivery vehicles, 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 (Berkner, K.L., Curr. Top. Micro. Immunol. 158:39-66. 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
  • PAVs have been designed to take advantage of the desirable features of adenovirus which render it a suitable vehicle for gene delivery.
  • adenoviral vectors can generally carry inserts of up to 8kb in size by the deletion of regions which are dispensable for viral growth, maximal carrying capacity can be achieved with the use of adenoviral vectors containing deletions of most viral coding sequences, including PAVs. See U.S. Patent No. 5,882,877 of Gregory et al.; Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731-5736, 1996; Parks et al., Proc. Natl Acad. Sci.
  • PAVs are deleted for most of the adenovirus genome
  • production of PAVs requires the furnishing 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 and can pose potential problems when administering the PAV to an individual if the contaminating helper adenovirus can replicate and be packaged into viral particles.
  • helper vector may contain mutations in the packaging sequence of its genome to prevent its packaging, an oversized adenoviral genome which cannot be packaged due to size constraints of the virion, or a packaging signal region with binding sequences that prevent access by packaging proteins to this signal which thereby prevents production of the helper virus.
  • 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, incorporated herein by reference).
  • Cre-Lox system Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996; Hardy et al., J. Virol. 71:1842-1849, 1997, incorporated herein by reference.
  • helper vectors reduce the yield of wild-type levels.
  • Another hurdle for PAV manufacturing aside from the problems with obtaining helper vector-free stocks, is that the production process is initiated by DNA transfections of the PAV genome and the helper genome into a suitable cell line, e.g., 293 cells. After cytopathic effects are observed in the culture indicating a successful infection, which may take up to from 2 to 6 days, the culture is harvested and is passaged onto a new culture of cells. This process is repeated for several additional passages, up to 7 times more, to obtain a modest cell lysate containing the PAV vector and any contaminating helper vector. See Parks et al., 1996, Proc. Natl Acad. Sci.
  • helper vectors which promote the production of substantially helper vector-free PAV stocks and there is a need for improved cell lines which simplify the PAV stock production process while being adaptable to large scale commercial production.
  • helper vectors and cell lines for the production of pseudoadenoviral (PAV) and other adenoviral vectors containing substantially reduced levels of contaminating helper vector.
  • the invention provides for helper vectors for the production of substantially helper vector-free PAV stocks comprising recombinase recognition sequences (e.g. Cre/Lox, Flp/FRT system, and phage [ ⁇ ] C31) which, depending upon their arrangement within the helper vector, can prevent helper vector packaging.
  • the recombinase recognition sequences employed are those from phage C31.
  • Phage C31 recombinase appears to be able to promote recombination/excision in a manner similar to Cre/Lox or Flp, but will not perform the reverse reaction to re-insert the excised piece. In theory, this recombinase should eliminate the generation of mutations and re-insertions that make the helper resistant to recombinase activity.
  • the invention also provides for improved cell lines for the production of substantially helper vector-free PAV stocks comprising a stably introduced novel circular PAV genome into the cell.
  • PAV adenoviral vectors from which a significant portion of the adenoviral genome has been deleted.
  • adenoviral vectors such as those described in WO94/12649 [Gregory et al]; WO99/64577
  • the functions of the deleted portion of the adenoviral genome may be supplied in trans, usually via co-infection with a replication incompetent helper virus, or by culturing the virus in a stock of transfected packaging cells.
  • the methods and materials of the present invention are useful for methods of producing recombinant adenoviral stocks with improved efficiency and safety characteristics.
  • the helper vector provides adenovirus (Ad) functions in trans and comprises an adenoviral genome containing recombinase recognition sequences placed in an inverted orientation relative to each other such that the action of the recombinase on the cognate recombinase recognition sequences inverts the central portion of the helper vector genome and displaces the packaging elements from their close proximity to both or either of the 5' and 3' ITR. This displacement results in the packaging elements being moved to a distance of approximately 3,000 nucleotides from the ITR. Since the distance from the ITR required for the proper functioning of the adenovirus (e.g.
  • Ad5 packaging motif is approximately 600 nucleotides from the ITR, such a recombination event compromises packaging of the helper vector while leaving intact the genes encoding replication functions and structural proteins necessary for the packaging of the PAV.
  • the recombinase recognition sequences in the helper vector are placed in the same orientation and at opposite ends of the helper genome. Action of the recombinase on the cognate recombinase recognition sequences leads to formation of a circular helper genome molecule and a separate short linear DNA that contains the left and right ITRs, i.e. the ITRs are excised entirely out of the genome.
  • helper vector comprises a packaging signal region flanked by recombinase (e.g.
  • Another object of the invention is to provide improved cell lines for the production of
  • Such improved cell lines comprise a circular PAV genome comprising a bacterial plasmid genome (e.g. pBR322), or an origin of replication and drug resistance sequences derived from a bacterial plasmid genome (e.g. pBR322) to allow propagation of the PAV in bacteria, and eukaryotic and or viral replication origin sequences (e.g. EB V sequences) to allow replication of the PAV genome within the mammalian producer cell line.
  • PAV production is initiated by infection of the cell line with a suitable helper vector, such as those described above comprising recombination recognition sequences for the prevention of helper vector production.
  • the helper vector provides, in trans, replication and packaging functions required for the production of PAV stocks.
  • the circular PAV genome is constructed such that there is a head-to-tail duplication of the ITR adjacent to the packaging sequence as would exist when Ad DNA is being replicated as a linear concatenate. See, e.g., Graham, 1984, EMBO J. 3:2917-2922; Fisher et al., 1996, Virol. 217: 11-22. Excision of the ITR from this configuration is more efficient than from a single ITR.
  • the circular PAV genome may comprise restriction endonuclease sites at positions just beyond the ITRs which, when excised by a restriction endonuclease, make the ITRs accessible and allow for efficient replication and packaging of the PAV.
  • the helper vector may provide nucleotide sequences encoding for the appropriate restriction endonuclease (e.g. RsrII, I-Ceu I, I-Ppo I; available from New England Biolabs, Beverly, MA).
  • Figure 1 shows a schematic diagram of an adenovirus helper vector whose genome, including a packaging signal, is flanked by two recombinase recognition sites placed in an inverted orientation relative to each other.
  • Figure 2 shows a schematic diagram of an adenovirus helper vector whose genome contains two recombinase recognition sites placed in the same orientation and at opposite ends of the helper genome.
  • FIG. 3 shows a schematic diagram of a circular pseudoadenoviral (PAV) vector comprising a bacterial plasmid genome for propagation of the PAV in bacteria and replication sequences from the EBV genome for replication in mammalian cell lines.
  • PAV circular pseudoadenoviral
  • Figure 4A shows a schematic diagram of a helper vector containing a packaging signal flanked by FRT sites and additionally containing hAAT stuffer sequences flanked by the FRT sites.
  • Figure 4B shows a schematic diagram of a helper vector containing a packaging signal flanked by Phage C31 sites and additionally containing hAAT stuffer sequences flanked by the Phage C31 sites.
  • Subconfluent 293 or 293/Flpe6 cells were infected with helper virus in a 6-well dish. Cells were harvested 3 days later and one tenth of the lysate was passaged onto subconfluent 293 or 293/Flpe6 cells. Cells were harvested 3 days later and the lysated subjected to titer analysis. Infected cells were monitored by expression of enhanced green fluorescent protein (EGFP)(Panel C).
  • EGFP enhanced green fluorescent protein
  • Figure 5 shows a schematic diagram of a PAV vector comprising the 5' ITR and packaging sequences of Ad2 (nucleic acids 1-356) separated by a multiple cloning site into which is inserted a nucleic acid encoding ⁇ gal.
  • Figure 6 shows a schematic diagram of a PAV vector comprising the 5' ITR and packaging sequences of Ad2 (nucleic acids 1-380) separated by a multiple cloning site into which is inserted a nucleic acid encoding ⁇ gal.
  • Figure 7 shows a schematic diagram of a PAV vector comprising the 5' ITR and packaging sequences of Ad2 (nucleic acids 1-356) separated by a multiple cloning site into which is inserted a nucleic acid encoding ⁇ gal and a nucleic acid encoding ⁇ gal.
  • Figure 8 A and 8B show particular embodiments of adenoviral helper genomes and PAV genomes useful in the present invention.
  • the invention is directed to novel adenoviral helper vectors 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.
  • PAV pseudoadenoviral vectors
  • the term PAV refers to adenoviral vectors derived from the genome of an adenovirus which contain minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can accommodate one or more transgenes, up to about 36 Kb of foreign nucleic acid, (see, e.g., disclosure of U.S. Patent No. 5,670,488, incorporated herein by reference).
  • the helper vectors of the invention are packaging defective due to the inclusion of recombinase recognition sequences in their genome which function to displace the viral packaging sequences from their necessary location in proximity to the ITRs.
  • the invention is also directed to PAV producer cell lines which are stably transfected with a PAV and which facilitate PAV production while being adaptable to large scale commercial production of PAV.
  • the invention is further directed to a PAV shuttle vector which can be propagated in bacteria and replicated in mammalian cells lines. Such a shuttle vector is useful for in vitro applications such as for the delivery of a nucleic acid to a cell for the production of proteins in vitro and for the in vitro study of proteins in a mammalian system.
  • the helper vectors, cell lines and PAV of the present invention are useful for the production of PAV with minimal contamination by helper vectors.
  • the present invention can be used with other replication incompetent adenoviral vectors.
  • adenoviral vectors include partially deleted adenoviral vectors, including those described in WO99/57296, the disclosure of which is hereby incorporated herein by reference.
  • PAV replication incompetent adenoviral vectors
  • helper vectors, cell lines and methods of the present invention will be designed to complement the specific configuration of the adenoviral genome present in the replication incompetent adenoviral vector.
  • Human adenovirus genomes comprise a double-stranded linear nucleic acid of approximately 36,000 bp, with a covalently attached terminal protein (TP) at each 5' terminus.
  • the 5' and 3' termini of adenoviruses are composed of inverted, repeated sequences ranging from 102 to 162 base pairs (Stillman, Cell 35:1-9, 1983). These inverted terminal repeats (ITRs) contain the origin of DNA replication and allow DNA synthesis to begin at either end of the molecule.
  • Packaging of the linear genome occurs in a polar fashion, from left to right, due to the location of the packaging element in the left end of the molecule.
  • the packaging element comprises a repeated motif, and it must reside near the end of the linear genome to function.
  • a helper vector 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 vector genome is disabled for packaging, thereby allowing for preferential packaging of PAV genomes into viral particles.
  • the helper vector 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 vector 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 vector genome As there may be very low levels of helper vector contamination of the final PAV preparation, it is advantageous to disable the helper vector genome by deletion of viral genes.
  • the E4 region may be deleted from the helper vector genome such that any helper vector that contaminates the final PAV preparation will be replication defective and not cause an adverse response in a subject.
  • the DNA polymerase gene may be deleted from the helper vector genome, such that any helper vector contamination of the final PAV preparation will also be replication defective.
  • the genes deleted from the helper vector genome will be stably placed within the PAV producer cell, e.g., the E4 or DNA polymerase genes noted above.
  • helper vector genome deletions make the helper vector progressively safe and lessen the negative consequences of helper vector contamination of the final PAV preparation.
  • the production of the proteins encoded by a helper vector genome facilitates the replication of the PAV genome during the production of PAV vector stock.
  • the adenovirus genes required in trans are not limited by virus serotype, and the helper vectors of the invention can contain adenovirus genes from more than one serotype.
  • Structural proteins, which are supplied by the helper vector can therefore be chosen so that the capsid proteins are derived from a desired serotype or serotypes and optimized for a particular use.
  • Ad6 adenovirus serotype 6
  • helper vector genome is desirably modified according to the present invention such that packaging of helper vector is impaired, or eliminated.
  • disability reduces or eliminates the production of helper vector in the preparation of a PAV stock, while allowing the helper vector itself to be propagated during the separate production of a helper vector stock.
  • a helper vector of the invention is rendered packaging-defective by incorporation into its genome, recombinase binding sequences which function to displace the packaging sequences from their necessary location in the helper vector genome in proximity to the ITRs, such displacement resulting in the inability to package.
  • a recombinase protein which may be supplied in the producer cell lines during production of PAVs and may bind to such recombinase binding sequences inserted into the helper vector genome.
  • a binding protein is defined as any protein or peptide which is capable of binding to the binding sequences inserted into the genome of the helper vectors of the present invention so as to prevent packaging or any protein or peptide which is capable of binding to and inducing cleavage at a particular binding sequence.
  • the binding protein is a recombinase which binds to specific nucleotide sequences.
  • the substantially helper vector-free PAV stocks of the present invention refer to PAV stocks preferably comprising less than about 5% helper vector.
  • Preferred specific binding sequences of the invention include, but are not limited to, those derived from (1) the FLP recombinase target site (FRT)
  • the packaging signal Upon recognition of the FRT, Lox or phage [ ⁇ ] C31 nucleotide sequences by either FRT, Lox or phage [ ⁇ ] C31 recombinase, the packaging signal is displaced from its position proximal to an ITR either by a recombination event which causes the packaging signal to be moved to another location in the helper vector genome, or by a recombination event which causes the excision of the ITR sequences from the helper vector genome.
  • Truncated and/or altered FRT, Lox or phage [ ⁇ ] C31 sites which bind FRT, Lox or phage [ ⁇ ] C31 respectively, are also within the scope of the present invention as well as any other sequences which facilitate the binding and recombination by FRT, Lox or phage [ ⁇ ] C31 recombinase.
  • Truncations may be made by deleting particular nucleotides and alterations may be made by mutating particular nucleotides by techniques known to those skilled in the art.
  • Truncated and or altered FRT, Lox or phage [ ⁇ ] C31 sites may be such that binding by FRT, Lox or phage [ ⁇ ] C31 recombinase is not compromised.
  • the invention also contemplates the use of other recombinase or integrase proteins and their respective DNA sequences to which they bind.
  • recombinase or integrase proteins There are currently approximately 105 proteins in subgroups of site specific recombinases. See generally, Nunes-Duby et al., 1998, Nucleic Acids Res. 26:391- 406; Argos et al, 1986, EMBOJ. 5:433-440.
  • Cre and FLP a recombinase (R) encoded by the pSRl plasmid of the yeast Zygosaccharomyces rouxii has similar function to FLP (Kilby et al., 1993, TIG 9:413-421).
  • the "R" recombinase and its recognition sequences may also facilitate the binding and recombination referred to herein.
  • the phage [ ⁇ ] C31 recombinase system is described in Kuhstoss and Rao, J. Mol Biol. 222:897-908 (1991); United States Patent 5,190,981; Groth et al., PNAS Early Edition, www.pnas.org/cgi/doi/10.1073/pnas.090527097; and PCT Patent Publication WOOO/l 1155. The disclosure of all of these publications is hereby incorporated herein by reference.
  • the FRT, lox, phage C31 attB, attP and other recognition and/or binding sites may be synthesized using standard techniques for oligonucleotide synthesis.
  • the nucleotide sequences which bind a recombinase or integrase protein are placed in an inverted orientation relative to each other; the left most sequence is placed approximately between nucleotide positions 102 and 194 of the helper vector genome ( Figure IB), and the right-most sequence is placed approximately between nucleotide positions 32,800 and 32,820 of the helper vector genome ( Figure IB).
  • Action of the recombinase on the cognate recombinase recognition nucleotide sequences inverts the central portion of the helper virus vector genome such that the packaging elements are displaced from their close proximity to the ITR and embedded within the right end of the helper genome at a distance of approximately 3,000 nucleotides from the ITR ( Figure 1C), a distance which significantly reduces the packaging of the helper virus vector.
  • Such reduction of packaging of the helper virus results in the production of a PAV stock which contains less than 5% contaminating helper vector.
  • the recombinase recognition sequences are placed in the same orientation at opposite ends of the helper vector genome; the left-most sequence placed approximately between nucleotide positions 102 and 194 ( Figure 2B), and the right-most sequence placed approximately between nucleotide positions 35,614 and 35,836 ( Figure 2B).
  • Action of the recombinase on the cognate recombinase recognition nucleotide sequences leads to formation of a circular helper genome molecule and a separate short, linear DNA that contains the left and right ITRs ( Figure 2C).
  • the circular helper cannot be packaged because the packaging sequence is no longer adjacent, or even on the same molecule, as the ITR.
  • helper genome may be accomplished through the use of alternative replication origins, such as those derived from SV40, EBV or a eukaryotic cell, preferably mammalian cells, which are incorporated into the helper vector genome (Figure 2C).
  • alternative replication origins such as those derived from SV40, EBV or a eukaryotic cell, preferably mammalian cells, which are incorporated into the helper vector genome ( Figure 2C).
  • Standard techniques of molecular biology such as restriction enzyme digestion and ligation, polymerase chain reaction and site-directed mutagenesis can be used to create a plasmid comprising the recombinase nucleotide binding sites of the present invention.
  • 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 helper vector, such that homologous recombination occurs, thereby generating a helper vector with the desired recombinase nucleotide binding sequence.
  • the resultant helper vector has exposed ITRs which are available to the adenovirus DNA polymerase and terminal protein for replication of the helper vector genome. See Figure IB and 2B.
  • helper vector in a cell line which does not provide for the recombinase protein and therefore does not support the recombination event which renders the helper vector packaging deficient or defective ( Figure 1C and 2C).
  • a single vector may be constructed which comprises the recombinase nucleotide binding sites and required sequences of the adenovirus helper genome.
  • the helper vector as depicted in Figure
  • the packaging signal region generally contains a minimum of seven AT- rich elements, denoted AI-AVII.
  • the helper virus genome may be modified by the deletion of packaging elements Al through ATV, retaining only the packaging elements AV, AVI, and AVII or a repeated (AV-AVI-AVII) 2 motif ( Figure 4).
  • Recombinase nucleotide binding sequences such as FRT or attB and attP sequences, may flank the packaging elements ( Figure 4; Helper 1).
  • hAAT stuffer sequences may be placed either 3 1 or 5' to the packaging elements and between the recombinase nucleotide binding sequences ( Figure 4; Helper 5 and Helper 6).
  • a stuffer sequence is a term generally recognized in the art intended to define functionally inert nucleotide sequence.
  • the stuffer sequence may be a nucleotide sequence which does not code for a protein and may be derived from intron sequences or other noncoding nucleotide sequences (e.g. a 450 bp DNA derived from intron sequences from human ⁇ -antitrypsin (hAAT); see Figure 4).
  • the stuffer sequences are useful to improve excision of the packaging elements from the helper vectors of the present invention.
  • Contaminating helper vector may be eliminated or greatly reduced in PAV stocks by techniques known in the art.
  • the helper vector is supplied to a cell line, e.g. 293 cells (Graham et al., J. Gen. Virol. 39:59-72, 1977), along with a packaging deficient PAV and a source for the recombinase protein (e.g.
  • the cell line may produce the recombinase protein
  • a vector may additionally be supplied encoding for the recombinase protein
  • the PAV may encode for the recombinase protein
  • the helper vector itself may have an inducible promoter driving the expression of the recombinase protein such that the recombinase would only be expressed during PAV production
  • PAV can be replicated and packaged by the necessary proteins provided in trans by the helper vector, while the helper vector itself cannot be packaged due to the rearrangement or excision of the ITR sequences such that packaging cannot proceed.
  • the PAV and the helper vector can contain signal regions or ITR sequences from different adenovirus serotypes such that sequence overlap between the PAV and the helper vector is minimized and the possibility of recombination is reduced.
  • the invention is further directed to PAV shuttle vectors comprising a bacterial plasmid genome, e.g. pBR322, or a bacterial origin of replication and drug selection sequences and a mammalian replication origin, such as replication origin sequences from the EBV genome or the SV40 genome (see figure 3).
  • a bacterial plasmid genome e.g. pBR322
  • a mammalian replication origin such as replication origin sequences from the EBV genome or the SV40 genome
  • the bacterial plasmid genome or bacterial replication origin and drug selection sequences allows for the production of the PAV in bacteria and the mammalian origin of replication allows for the replication of the PAV in mammalian cells.
  • the PAV vector may comprise a transgene and a nucleic acid encoding EBNA or S V40 large T antigen, alternatively, the nucleic acid encoding the EBNA or SV40 large T antigen may be placed within the genome of the mammalian producer cell line or on a separate plasmid.
  • the PAV is circular ( Figure 3), making the ITRs embedded rather than free, thereby reducing the efficiency of replication and packaging of the PAV, as compared to a PAV with free ITRs.
  • a process that is known to occur (Graham, 1984, EMBO 3:2917-2922; Matani et al, 1995, Proc. Natl. Acad. Sci.
  • the PAV ITRs may be flanked by a restriction endonuclease nucleotide binding site which, when cleaved by a restriction enzyme, e.g. RsrII, free the ITRs to promote efficiency of replication and packaging of the PAV.
  • a restriction enzyme e.g. RsrII
  • the PAV vectors of the present invention comprise two ITR sequences and the minimal adenovirus 5' sequences required for helper vector-dependant replication and packaging of the PAV. See U.S.
  • Patent Application 5,882,877 incorporated herein by reference.
  • the PAVs of the present invention contain no potentially harmful viral genes, have a capacity for foreign nucleic acids of approximately 36 kb, may be produced in reasonably high titers and maintain the tropism of the parent virus for dividing and non-dividing target cell types. See U.S. Patent Application 5,882,877.
  • the invention is also directed to methods for the production of PAVs and other deleted adenoviral vectors in high yield, using the helper vectors, PAVs and producer cell lines of the present invention.
  • PAV is preferentially produced, generating an enriched preparation which is substantially free of contaminating helper vector.
  • 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 to expression control sequences, can be engineered as a circular PAV and can be stably introduced into a cell to provide an improved cell line for the production of PAV.
  • PAV can be delivered to cells by any method of nucleic acid transfer, including, but not limited to, transfection, lipofection and electroporation to produce the PAV-transfected cells of the invention.
  • helper vector can be provided to the PAV-transfected cell line to initiate PAV production thus eliminating the need for multiple rounds of infection and reducing the time and effort required to produce a PAV stock.
  • 2 to 20 ⁇ g of PAV DNA is delivered to a cell by lipofection using lipofectamine (Gibco BRL, Rockville, Md) or a CaPO 4 kit such as Profectin (Promega, Madison, WI), and the cells are infected with a helper vector of the invention using a multiplicity of infection (MOI) of 0.5 to 10.
  • lipofectamine Gibco BRL, Rockville, Md
  • CaPO 4 kit such as Profectin (Promega, Madison, WI)
  • MOI multiplicity of infection
  • the improved helper vectors comprise recombinase nucleotide binding sites which either excise or rearrange the ITR sequences of the helper vector to reduce or eliminate helper vector contamination of the PAV stock.
  • the reduction or elimination of helper vector contamination eliminates or reduces the need for purification procedures to remove contaminating helper vector.
  • the improved cell lines and helper vectors are amenable to adaptation to large scale commercial production due to the simplification and elimination of the need for lengthy production and purification protocols.
  • the helper vectors comprise phage [ ⁇ ] C31 nucleotide binding sites attB and attP located such that they will either excise, rearrange or otherwise deactivate the ITR sequences of the helper vector to reduce or eliminate helper vector contamination of the PAV stock, for example by preventing packaging from occurring.
  • the phage [ ⁇ ] C31 recombinase offers particular advantages for the methods of the present invention in that the excision reaction is essentially a one way reaction; very little, if any of the reverse process [integration] will occur. Thus, the reappearance of helper vector which can be packaged and produce contaminating helper virus is minimized.
  • phage [ ⁇ ] C31 sequences minimally required for use as recognition sites have recently been identified, the length of these sequences, attB and attP, being approximately 34 and 39 nucleotides, respectively [Groth et al., PNAS Early Edition, supra].
  • the phage [ ⁇ ] C31 recombinase system may be used in practical applications to viral vector production, as described herein.
  • phage C31 recognition sites are employed.
  • purification of the PAV from the 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 standard CsCl gradients.
  • the PAV particles are of lower density relative to the helper particles 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 WO 97/08298, 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.
  • Preferred transgenes for use in the methods of the present invention include hemophilic factors, preferably Factor Vila [US 4,784,950]; Factor Vffl [US 4,965,199; US 4,868,112 [B- domain deleted] and and US 5,661,008]; and Factor IX [US 4,994,371].
  • transgenes are those which encode lysosomal storage enzymes, including genes encoding glucocerebrosidase [Gaucher's disease; US 5,879,680; US 5,236,838]; alpha-galactosidase [Fabry Disease; US 5,401,650]; acid alpha-glucosidase [Pompe's Disease; WO00/12740]; alpha- n-acetylgalactosaminidase [Schindler Disease; US 5,382,524]; acid sphingomyelinase [Niemann- Pick disease; US 5,686,240]; alpha-iduronidase [WO9310244A1].
  • transgenes include the genes for CFTR, dystrophin and alpha-antitrypsin.
  • Transgene activity of a PAV preparation may be monitored by immunoflourescent techniques by infecting 293 cells with the PAV, then using an antibody against a PAV-encoded transgene expression product (protein) to determine infectious particles.
  • the activity of an enzyme encoded by a transgene can be measured (e.g. ⁇ -galactosidase, ⁇ - galactosidaseA, ⁇ -antitrypsin), by, for example, an ELISA assay.
  • transgene is CFTR
  • expression of a CFTR transgene in target cells from a subject with cystic fibrosis is correlated with production of a functional chloride ion channel in such cells that may be measured by techniques known in the art.
  • the transgene is ⁇ -galactosidase
  • its expression in target cells from an individual with Fabry's disease can be monitored by a decrease in stored lipid substrate, GL3, see, e.g. , PCT/US98/22886, filed 10.29.98, incorporated herein by reference.
  • the ability of PAV to enter cells may be 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 flourescent in situ hybridization (FISH). Minimal contamination of the PAV stock is expected using the novel helper viruses of the invention. Helper virus production, if any, can be scored, for example, by standard plaque assays on 293 cells. Preferably, the ratio of PAV to helper vector will be greater than 10,000 to 1.
  • Flp is a recombinase derived from yeast with an optimal reaction temperature of 30° C (Buchholz et al., 1998, Nature Biotechnol. 16:657-662). To make this enzyme more useful when expressed in mammalian cells, a thermal resistant form of the enzyme has been engineered (Buchholz et al., 1996, NAR 24:4256-4262); this thermal resistant form of Flp is known as Flpe6. Mammalian cells in which the action of this enzyme is used for PAV packaging include 293 cells (Graham, F . L . , J . Gen .
  • Flpe6 enzyme Expression of the Flpe6 enzyme is achieved by cloning the Flpe ⁇ coding sequence into the expression cassette within the plasmid pCEP4 (Invitrogen, Carlsbad, CA) which contains the CMV promoter and the gene encoding hygromycin drug resistance.
  • An alternate expression system for Flpe ⁇ is obtained by cloning the Flpe ⁇ coding sequence into the expression cassette in the plasmid pTRE which contains a minimal promoter regulated by the tetracycline gene control system. Gossen and Bujard, Proc. Natl Acad. Sci.
  • TetR tetracycline repressor
  • VP16 herpes virus VP16 transcriptional activation domain
  • TetR VP 16 and rTetR/vpl ⁇ controls the expression of genes linked to a minimal promoter cloned adjacent to tetracycline transcriptional regulatory elements (TRE).
  • TRE tetracycline transcriptional regulatory elements
  • PERC6 Flpe6 cell line PERC6 cells (Fallaux et al., Human Gene Therapy 9: 1909- 17 (1998)) were plated at 7.8 x 10 6 cells/10 cm dish and transfected with 10 ⁇ g of pCEP-4/Flpe ⁇ plasmid using 60 ⁇ l of lipofectamine. Three days post transfection, plates were split to 15 cm plates and drug selection was applied (150 ⁇ g/ml hygromycin). Media was changed 2-3 times per week until colonies were picked 3 weeks post transfection. Cell clones were then put under maintenance dosage of hygromycin (100 ⁇ g/ml).
  • 293 Tet-off/Flpe6 cell line 293 Tet-Off cells were seeded into Biocoat Collagen 1 10 cm 2 dishes (Becton Dickinson, Bedford, MA) at 6 X 10 5 cells per dish the day before transfection. The next day, medium was removed from the plates and replaced with -10 ml of fresh medium three hours prior to time of transfection. Approximately 45 minutes before transfection, a Calcium Phosphate transfection mixture was prepared using the desired plasmid DNA according to the Promega Profectin Mammalian transfection protocol. Cells were transfected with 10 ⁇ g of pTRE/Flpe ⁇ DNA and 2 ⁇ g of pTK Hygromycin resistance marker DNA.
  • the transfection mixture was allowed to precipitate for half an hour before being added drop-wise to cells on plates. The next day, the plates were washed with fresh medium and re-fed. Two days later, the plates were harvested and plated out to 150 mm dishes. Twelve plates were seeded at 2000-2500 cells per plate.
  • the DNA cloning plates receive -MEM medium containing 15% Tet System Approved FBS, 2 ⁇ g/ml of Tetracycline, 2 mM L-Glutamine, 100 ⁇ g/ml of G418, and 50 ⁇ g/ml of Hygromycin for selection. Plates were incubated for a two week period, and were inspected for colonies of drug resistant cells. Hygromycin resistant clones which were isolated using 8 cm cloning rings and silicon grease.
  • Example 2 Improved Helper Vector Where the Packaging is Impaired Due to Repositioning of the Packaging Sequence to an Internal Position in the Helper Genome
  • the nucleotide sequences which bind the Flp recombinase protein are placed in an inverted orientation relative to each other.
  • the recognition sequence for the Flp recombinase is referred to as FRT and has the following sequence:
  • helper vector genome When this helper vector genome is introduced into the Flp-expressing producer cell, action of the Flp recombinase on these FRT sequences inverts the central portion of the helper vector genome such that the packaging elements are displaced from their close proximity to the ITR and embedded within the helper genome at a distance of approximately 3,000 nucleotides from the ITR ( Figure 1C), a distance which reduces the packaging of the helper virus vector such that there is less than 5% contamination of the PAV preparation by the helper vector.
  • the FRT sequences are prepared as synthetic oligonucleotides and are inserted into the desired positions within the helper genome using standard molecular cloning methods.
  • the helper vector genome cloned as a plasmid bearing the entire helper genome (Souza and Armentano, 1999, Biotechniques 26:502-508), is converted to an infectious virus by transfection into 293 cells or PERC6 cells (0.5 to 10 micrograms of DNA) using standard calcium phosphate or lipofectamine.
  • helper virus genome is cloned into two or more DNA fragments, each with overlapping segments of the helper genome such that recombination between the separate helper DNA fragments occurs after transfection into 293 or PERC6 cells, resulting in the generation of infectious helper virus.
  • Evidence of viral infection is noted by generalized cytopathic effect or the presence of distinct viral plaques.
  • the helper virus is recovered by passaging on 293 cells to provide a helper virus stock.
  • Example 3 Improved Helper Vector Where the Helper Vector is Converted to an Episomal Form Lacking ITRs During PAV Production
  • the nucleotide sequences which bind the Flp recombinase protein are placed in the same orientation relative to each other.
  • the recognition sequence for the Flp recombinase is referred to as FRT and has the following sequence: GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC (SEQ ID NO:l).
  • the left most sequence is placed such that the 5' G nucleotide of the FRT sequence follows nucleotide position 102 of the helper vector genome ( Figure IB), and the 5' G of the FRT sequence follows nucleotide positions 35,835 in one helper version, following nucleotide position 35,850 in a second helper version, and following nucleotide position 35,875 in a third helper version. ( Figure IB).
  • helper genomes containing FRT sequences When any of these helper genomes containing FRT sequences is introduced into the Flp-expressing producer cell, action of the Flp recombinase on the FRT sequences converts the linear helper genome to a circular episomal form which now lacks the ITRs. Because the packaging sequence is now separated from the ITRs, the helper genome cannot be packaged. Additionally, the fact that the packaging sequence in buried within the helper genome further prevents packaging.
  • the helper with this arrangement of FRT sequences yields less than 5% contamination of a PAV stock preparation by the helper vector. Replication of the episomal helper genome is driven by the inclusion of oriP sequences from the EBV genome.
  • sequences are isolated from the plasmid pCEP-4 (Invitrogen) by PCR or enzyme digestion and are inserted into the helper genome at nucleotide position (27,973-30,937 to replace the E3 region; ⁇ 2.9). Replication from oriP is driven by the action of the EBNA gene from EBV.
  • the gene for EBNA is included in the plasmid pCEP-4 (Invitrogen), and in introduced into the cell by standard transfection methods.
  • the FRT sequences are prepared as synthetic oligonucleotides and are inserted into the desired positions within the helper genome using standard molecular cloning methods.
  • helper genome cloned as a plasmid bearing the entire helper genome is converted to an infectious virus by transfection into 293 cells or PERC6 cells (0.5 to 10 micrograms of DNA) using standard calcium phosphate or lipofectamine.
  • the helper virus genome is cloned into two or more DNA fragments, each with overlapping segments of the helper genome such that recombination between the separate helper DNA fragments occurs after transfection into 293 or PERC6 cells, resulting in the generation of infectious helper virus.
  • Evidence of viral infection is noted by generalized cytopathic effect or the presence of distinct viral plaques.
  • the helper virus is recovered by passaging on 293 cells to provide a helper virus stock.
  • Mammalian cells appropriate for bearing episomal PAV genomes include 293 cells, PERC6 cells, HeLa cells, A549 cells, as well as variants of these cells that have been altered by the introduction of the tetracycline gene expression control system (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA 89:5547; Gossen et al., 1995, Science 268:1766 ' .. Flpe ⁇ sequences are incorporated into the cells as described above. Circular bacterial plasmids containing an entire Ad genome, their construction, and manipulation have been described (Souza and Armentano, 1999, Biotechniques 26:502-508).
  • the Ad sequences that are retained are those required for PAV (U.S. Patent No. 5,882,877, issued March 16, 1999, incorporated herein by reference), the ITR sequences and packaging sequences.
  • the remainder of the PAV genome comprises the transgene of interest and stuffer sequences as may be required. (e.g. PAV356- ⁇ gal, PAV380- ⁇ gal, PAV380- ⁇ al Figures 5, 6 and 7 respectively).
  • the oriP and EBNA genes from EBV are cloned as well as the drug resistance gene (e.g., hygromycin, G418, blasticidin) to provide a means of maintaining the episomal PAV within the cell.
  • a variant of the episomal PAV genome is constructed where an additional ITR is placed directly adjacent to the left ITR in a head to tail fashion.
  • Excision of the left end of the PAV genome occurs when the cell is infected with a PAV helper, and packaging of the PAV into virions ensues.
  • the rate of excision is increased from the head to tail left-hand ITR configuration of episomal PAV.
  • Excision of the left end of the PAV episome is also enhanced by incorporating recognition sequences for restriction endonucleases at positions immediately flanking the outer limits of the ITRs.
  • Appropriate restriction endonucleases for this purpose include Rsrll, I-Ceul, I-Ppol, and others for which there are no recognition sequences within the helper genome or the PAV genome.
  • the Phi C31 integrase cDNA was PCR amplified from phi C31 DNA (gift Keith Chater, John Innes Center, England) in a 50 uL reaction volume containing IX Pfu buffer (Stratagene, La Jolla, CA), 100 ng forward primer phi-1 (5'- ACC GCT CGA GAT GAC ACA AGG GGT TGT GAC C)[SEQ ID NO:7] and 100 ng reverse primer phi-2 (5'- GTC GAC TAG TCT ACG CCG CTA CGT CTT CCG T)[SEQ ID NO:8] and 5 units of Pfu polymerase.
  • IX Pfu buffer (Stratagene, La Jolla, CA)
  • 100 ng forward primer phi-1 5'- ACC GCT CGA GAT GAC ACA AGG GGT TGT GAC C
  • 100 ng reverse primer phi-2 5'- GTC GAC TAG TCT ACG CCG CTA CGT CTT CCG T
  • the reaction was subjected to 35 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes.
  • the 1843 bp product was subcloned into the Xho I and Spe I site of vector pCMV to create pCMVint and completely sequenced.
  • a previral target plasmid containing two copies of Ad2 packaging elements (AV-AVI- AVII) flanked by ⁇ ttP and ⁇ ttB attachment sites was constructed by annealing oligos att-1 (5'- CTA GTC CCC AAC TGG GGT AAC CTT TGA GTT CTC TCA GTT GGG GGC GCG TAA TATTTG TCTAGG GCC GCG GGGACT TTG ACC GTT TAC GTG GAGACT CGC G) [SEQ IDNO:9], att-2 (5'-CTC CAC GTA AAC GGT CAAAGT CCC CGC GGC CCTAGA CAAATA TTA CGC GCC CCC AAC TGA GAG AAC TCAAAG GTTACC CCA GTT GGG GA)[SEQ IDNO.10], att-3 (5'-TAA TAT TTG TCTAGG GCC GCG GGGACT TTGACC GTTTAC GTG GAG ACT GCTAGC GTG CCA GGG CGT GCC
  • Reticuloyte Lysate System Promega, Madison, WI. Briefly, 1 ⁇ g of pCMVint plasmid DNA was mix with TnT Lysate Reaction components in the presense of 35 S-methionine according to the manufacturer's instructions. Reactions were incubated for 2 hr at 37°C and stopped by adding 0.1 volumes SDS sample buffer. Five microliters was heat denatured at 95°C for 2 minutes and elecfrophoresed through a 4 - 15% SDS PAGE gel (Bio-Rad, Hercules, CA) for 1 hr at 125 volts. The gel was dried under vaccuum at 80°C and exposed to autoradiography film for 1 hr. A band of- 85 kd corresponding to the phi C31 integrase was present. Integration assay
  • PCR was performed using 5 ⁇ L DNA as template, 100 ng forward primer 2900LC (5'- GTG ACG TAG TAG TGT GGC)[SEQ ID NO: 13], 100 ng reverse primer 2876LC (5'- ACA CTA CGA CCT ACA CTG G)[SEQ ID NO: 14], IX Taq polymerase buffer (Promega, Madison, WI), 200 nM each dNTP, and 5 units Taq polymerase (Promega). Reactions were cycled 35 times at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes. Products were separated on a 2% super fine resolution agarose gel (Amresco, Solon, OH).
  • Expected sizes are 399 bp for unintegrated products which retain the Ad2 packaging sequence and - 270 bp for products deleted of the packaging sequence by integration between att? and ⁇ ttB sites.
  • a partially deleted adenoviral helper virus is employed in which the adenoviral protein IX relocated and most of the E4 region deleted, except for retaining the E4ORF6 region.
  • adenoviral vectors are described, for example, at US Patent 6,093,567, the disclosure of which is hereby incorporated herein by reference.
  • the packaging signal of the helper virus is flanked by attB and attP recognition sites to allow for disabling packaging of the helper virus.
  • the packaging cell line for the adenovirus is transfected with a phage C31 integrase plasmid or virus comprising a promoter operably linked to a DNA coding sequence encoding the phage c31 integrase, and an operably linked polyadenylation [PA] sequence.
  • the phage C31 integrase plasmid may be stably incorporated into a cell line, such as a 293-based cell line or PERC6.
  • the promoter is preferably a constitutive promoter which is well-expressed in the packaging cell, such as the cytomegalovirus promoter [CMV], or an inducible promoter such as the ecdysone promoter.
  • the polyadenylation sequence may be any appropriate PA sequence, such as the SV40 PA.
  • Example 6 Experiment with increasing helper contamination upon passaging Several PAV amplifications with performed using three different helpers.
  • Lysate harvested 7 days post infection had a helper titer of 6.4E5 by hexon staining. Four mis of this lysate was used to infect 1E6 cells/ ⁇ cm plate. Lysate harvested 5 days post infection had a helper titer of 1.3E7 IU/ml by EGFP staining. 2.5mls of this lysate was used to infect 1E6 cells/6cm plate. Lysate harvested 2 days post infection had a helper titer ⁇ 1.4E7 IU/ml by hexon staining. 3.5mls of this lysate was used to infect 3E6 cells/lOcm plate.
  • Lysate harvested 4 days post infection had a helper titer of 3.4E7 by hexon staining. 8.5 mis of this lysate was used to infect lE7cell/15cm plate. The harvested lysate had a titer of 4E7
  • Hirt DNA was isolated. PCR was done on the Hirt DNA using primers flanking the FRT sites. The PCR products corresponding to the unflipped product were cloned into the pCR2.1-TOPO vector and sequenced. Preparation of Hirt DNA:
  • Cells were lysed in lOmM Tris, lOmM EDTA and 0.6%SDS. Following the addition of 1M NaCl, the lysate was pronase-treated and cleaned up by phenol/chloroform.

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Abstract

La présente invention concerne des vecteurs auxiliaires améliorés ainsi que des lignées cellulaires destinées à la production de vecteurs pseudoadénoviraux (PAV) contenant des niveaux sensiblement réduits de vecteur auxiliaire contaminant. L'invention concerne des vecteurs auxiliaires destinés à la production de populations de PAV sensiblement exemptes de vecteurs auxiliaires comprenant des séquences de reconnaissance de recombinase phag C31 lesquelles, selon leur arrangement à l'intérieur du vecteur auxiliaire, peuvent empêcher l'encapsidation de vecteurs auxiliaires. L'invention concerne également des lignées cellulaires améliorées destinées à la production de populations de PAV, sensiblement exemptes de vecteurs auxiliaires comprenant un génome PAV circulaire introduit de manière stable dans la cellule.
PCT/US2000/025131 1999-09-23 2000-09-14 Vecteurs auxiliaires et lignees cellulaires de production de vecteurs pseudoadenoviraux WO2001021824A1 (fr)

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WO2007125146A1 (fr) * 2006-04-28 2007-11-08 Universitat Autonoma De Barcelona Procédé de production de vecteurs adénovirus destinés a la thérapie génique et séquences d'adn utilisées à ces fins
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EP1379281A2 (fr) * 2001-03-26 2004-01-14 The Board Of Trustees Of The Leland Stanford Junior University Systeme de vecteurs adenoviraux dependants d'un auxiliaire, et methodes d'utilisation
EP1379281A4 (fr) * 2001-03-26 2005-05-18 Univ Leland Stanford Junior Systeme de vecteurs adenoviraux dependants d'un auxiliaire, et methodes d'utilisation
US6943012B2 (en) 2001-03-26 2005-09-13 The Board Of Trustees Of The Leland Stanford Junor University Helper dependent adenoviral vector system and methods for using the same
WO2007125146A1 (fr) * 2006-04-28 2007-11-08 Universitat Autonoma De Barcelona Procédé de production de vecteurs adénovirus destinés a la thérapie génique et séquences d'adn utilisées à ces fins
WO2022069523A1 (fr) * 2020-09-29 2022-04-07 Albert-Ludwigs-Universität Freiburg Sauvetage d'adénovirus recombinés par résolution terminale in vivo médiée par crispr/cas
CN114574523A (zh) * 2022-03-14 2022-06-03 济南宜明医疗科技有限公司 用于aav病毒包装用的辅助质粒载体、其构建方法和应用

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