EP1025251A1 - Cassettes d'encapsidation de virus adeno-associe (aav) amplifiable pour la production de vecteurs de aav recombines - Google Patents

Cassettes d'encapsidation de virus adeno-associe (aav) amplifiable pour la production de vecteurs de aav recombines

Info

Publication number
EP1025251A1
EP1025251A1 EP98953640A EP98953640A EP1025251A1 EP 1025251 A1 EP1025251 A1 EP 1025251A1 EP 98953640 A EP98953640 A EP 98953640A EP 98953640 A EP98953640 A EP 98953640A EP 1025251 A1 EP1025251 A1 EP 1025251A1
Authority
EP
European Patent Office
Prior art keywords
aav
packaging
rep
gene
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98953640A
Other languages
German (de)
English (en)
Inventor
Carmel M. Lynch
Haim Burstein
Anthony M. Stepan
Dara H. Lockert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ampliphi Biosciences Corp
Original Assignee
Targeted Genetics Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Targeted Genetics Corp filed Critical Targeted Genetics Corp
Publication of EP1025251A1 publication Critical patent/EP1025251A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • a sequence at the AAV integration site shares homology with the AAV inverted terminal repeat (ITR) sequence, exhibits activity in a cell-free replication system, and is believed to be involved in both the integration and rescue of AAV. See, e.g., Weitzman et al., id., Kotin et al. (1992) EMBO J. 11 :5071-5078, and Urcelay et al., J. Virol. 69: 2038-2046.
  • ITR A sequence at the AAV integration site
  • the Rep proteins primarily Rep78 and Rep68, also exhibit pleiotropic regulatory activities including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894; Labow et al., 1987, Mol. Cell. Biol, 7:1320-1325; Khleif et al, 1991, Virology, 181:738-741).
  • the AAV p5 promoter is negatively auto- regulated by Rep78 or Rep68 (Tratschin et al, 1986, Mol. Cell. Biol. 6:2884-2894).
  • the capsid proteins VP1, VP2, and VP3 share a common overlapping sequence, but VP1 and VP2 contain additional amino terminal sequences. All three proteins are encoded by the. same cap gene reading frame typically expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 can be generated from this mRNA by use of alternate initiation codons. Generally, transcription from p40 yields a 2.6 kb precursor mRNA which can be spliced at alternative sites to yield two different transcripts of about 2.3 kb. VP2 and VP3 can be encoded by either transcript (using either of the two initiation sites), whereas VP1 is encoded by only one of the transcripts.
  • replication origins such as those present in eukaryotic or prokaryotic chromosomes, viral genomes, organelle genomes, and bacteriophage genomes, for example, and other origin-like or "or/ ' -like" sequences can be used in the practice of the invention (e.g., as alternatives or additions to the use of PI).
  • origin-like or "or/ ' -like" sequences can be used in the practice of the invention (e.g., as alternatives or additions to the use of PI).
  • activatable origins are those that are not constitutive, but rather require a signal before replication initiation and subsequent amplification of linked sequences will occur.
  • transcriptional regulatory sequence refers to a nucleotide sequence that controls the transcription of a gene or coding sequence to which it is operably linked.
  • Transcriptional regulatory sequences of use in the present invention generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.
  • a “recombinant AAV vector” refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or “transgenes” that are flanked by AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus and that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • AAV Rep and Cap proteins i.e. AAV Rep and Cap proteins
  • the rAAV vector in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector is typically referred to as a "pro- vector" which can be "rescued” by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.
  • Effective when used in describing a cell line refers to certain useful attributes of the line; in particular, the growth rate, and (for packaging cell lines) the number of viras particles produced per cell.
  • "Efficient growth” of a packaging cell line refers to the effective growth rate of the packaging cell, related to a comparable parental-type cell (i.e., a cell that does not carry an introduced AAV packaging gene)
  • the relative growth rate is at least 20% of the parental type, more preferably, 40%, more preferably, 80%), still more preferably, 90% and, most preferably, 100%.
  • “High efficiency packaging” indicates production of at least about 100 viral particles per cell, more preferably at least about 1,000 viral particles per cell, still more preferably at least about
  • These cells can be used as recipients for recombinant vectors, virases or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.
  • the methods and compositions of the invention will therefore utilize recombinant DNA constracts wherein AAV packaging genes are amplifiably linked to one or more activating elements.
  • the presently preferred activating elements are exemplified by PI and PI -like elements that exhibit structural and functional properties related to initiation of replication. Most preferred are elements that act as helper function-inducible origins of replication. Other sequences that can be directly or indirectly induced to initiate replication in response to helper function will also be useful in the practice of the invention. In addition, sequences that are not inducible by helper function, but which can be induced to initiate DNA replication by other stimuli (provided and/or controlled by the user), are also useful as activating elements in the practice of the invention.
  • This pFR400 fragment was comprised of a modified DHFR gene, with a reduced affinity for methotrexate (Mtx), transcriptionally linked to the SV40 early promoter and followed by the polyadenylation site from the Hepatitis B viras (HBV) surface antigen gene.
  • the pFR400 fragment was prepared by first digesting with Sail, followed by a four base pair fill-in (to generate a blunt end) and subsequent PvuII digestion and gel purification.
  • the resulting construct, p5repcapDHFR ( Figure 1), contains a DHFR gene whose transcription is regulated by an upstream SV40 early promoter and a downstream Hepatitis B Viras polyadenylation site. Immediately downstream of this DHFR transcriptional cassette lie the AAV rep and cap genes, followed by an AAV polyadenylation site.
  • DMEM complete After electroporation, cells were resuspended in DMEM complete (see Example A 7) and allowed to recover at 37°C in a humidified atmosphere of 10% CO 2 . After 24 hours, cells were subjected to selection in complete medium containing 500 nM methotrexate.
  • Clonal cell lines were derived from the PIRCD polyclonal population by limiting dilution. Producer lines are generated by introduction of an rAAV vector constract into a clonal PI RCD-containing packaging line .
  • Example A 8 Total genomic DNA prepared and digested according to Example A 9 for polyclonal samples PIRCD and p5repcapDHFR (Example A 8) was analyzed by the Southern blotting method of Example A 10. Degree of amplification was measured by relative photon intensity of the 3.8 kb band determined from phosphorimaging according to Example A 10. DNA from PIRCD-containing cells gave a value of 406,725 intensity units for the 3.8 kb band, while DNA from cells containing p5repcapDHFR gave a value of 30,211. Thus the presence of PI, in the PIRCD polyclonal line, is responsible for a 13.5- fold amplification of rep and cap genes, in the presence of adenoviras.
  • the rAAVCFTR or ACAPSN vector was transfected into HeLa cells via electroporation. Individual clones were isolated and screened for an intact, stably integrated rAAV vector.
  • the Pl/p5repcap/Rep8 packaging cassette was then transfected into HeE AAV-CFTR cells via Ca ⁇ PO 4 -mediated transfection and stable transfectants were selected using 2.5 mM L-histidinol.
  • a Hei ⁇ AAV-CFTR cell line containing a p5repcap(-Pl)/Rep8 packaging cassette was generated in similar fashion.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des constructions d'encapsidation d'AAV de haute efficacité ainsi que leurs méthodes d'utilisation. Ces constructions d'encapsidation de haute efficacité comprennent un élément d'activation (tel que la séquence P1 située à l'intérieur du site d'intégration AAV-S1 du chromosome humain 19), lié de manière amplifiable à un ou à un plusieurs gènes d'encapsidation d'AAV. Les constructions peuvent être soit intégrées dans le génome d'une cellule mammifère soit maintenues de manière épisomique. L'utilisation des vecteurs d'encapsidation d'AAV de haute efficacité de l'invention permet la production amplifiable et régulée de constructions de vecteurs de AAVr.
EP98953640A 1997-10-21 1998-10-20 Cassettes d'encapsidation de virus adeno-associe (aav) amplifiable pour la production de vecteurs de aav recombines Withdrawn EP1025251A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US955232 1978-10-27
US95523297A 1997-10-21 1997-10-21
PCT/US1998/021938 WO1999020779A1 (fr) 1997-10-21 1998-10-20 Cassettes d'encapsidation de virus adeno-associe (aav) amplifiable pour la production de vecteurs de aav recombines

Publications (1)

Publication Number Publication Date
EP1025251A1 true EP1025251A1 (fr) 2000-08-09

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EP98953640A Withdrawn EP1025251A1 (fr) 1997-10-21 1998-10-20 Cassettes d'encapsidation de virus adeno-associe (aav) amplifiable pour la production de vecteurs de aav recombines

Country Status (5)

Country Link
EP (1) EP1025251A1 (fr)
JP (1) JP2001520051A (fr)
AU (1) AU758541B2 (fr)
CA (1) CA2308008A1 (fr)
WO (1) WO1999020779A1 (fr)

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US6924128B2 (en) 1994-12-06 2005-08-02 Targeted Genetics Corporation Packaging cell lines for generation of high titers of recombinant AAV vectors
US6989264B2 (en) 1997-09-05 2006-01-24 Targeted Genetics Corporation Methods for generating high titer helper-free preparations of released recombinant AAV vectors
WO2000009693A2 (fr) 1998-08-11 2000-02-24 Darwin Discovery Ltd. Identification du gene responsable du phenotype de la souris 'scurfy' et de son orthologue humain
US6893865B1 (en) * 1999-04-28 2005-05-17 Targeted Genetics Corporation Methods, compositions, and cells for encapsidating recombinant vectors in AAV particles
AU783037B2 (en) 1999-05-28 2005-09-15 Targeted Genetics Corporation Methods and compositions for lowering the level of tumor necrosis factor (TNF) in the TNF-associated disorders
EP1939300A1 (fr) 1999-05-28 2008-07-02 Targeted Genetics Corporation Procédés et compositions pour diminuer le niveau de facteur de nécrose de tumeur (TNF) pour des troubles associés au TNF
DK1916258T3 (da) 1999-08-09 2014-07-28 Genzyme Corp Forøgelse af ekspression af en enkeltstrenget, heterolog nukleotidsekvens fra rekombinante, virale vektorer ved en sådan udformning af sekvensen at den danner intrastrengbasepar
EP1916258B1 (fr) 1999-08-09 2014-04-23 Targeted Genetics Corporation Améliorations de l'expression d'une séquence de nucléotides hétérologues à brin unique à partir de vecteurs viraux recombinants par la désignation de la séquence de manière à ce qu'elle forme des paires de bases intrabrins
DE10066104A1 (de) * 2000-09-08 2003-01-09 Medigene Ag Wirtszellen zur Verpackung von rekombinantem Adeno-assoziiertem Virus (rAAV), Verfahren zu ihrer Herstellung und deren Verwendung
JP4353701B2 (ja) 2001-05-08 2009-10-28 ダーウィン モレキュラー コーポレイション Ｆｏｘｐ３蛋白質を用いた霊長類における免疫機能の調節方法
ES2686504T3 (es) 2009-05-02 2018-10-18 Genzyme Corporation Terapia génica para trastornos neurodegenerativos
CN104603272B (zh) * 2012-07-06 2017-11-07 宝生物工程株式会社 能够产生腺伴随病毒载体的细胞

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US5173414A (en) * 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
JPH09509564A (ja) * 1993-11-09 1997-09-30 ターゲテッド ジェネティックス コーポレイション 高力価組換えａａｖベクターの生成
US6207457B1 (en) * 1995-09-08 2001-03-27 Avigen, Inc. Targeted nucleotide sequence delivery and integration system
ES2317646T3 (es) * 1995-09-08 2009-04-16 Genzyme Corporation Vectores aav mejorados para terapia genica.
JP2002514899A (ja) * 1996-03-04 2002-05-21 ターゲティッド ジェネティックス コーポレイション 組換えａａｖベクターを用いて血管中の細胞を形質導入するための方法

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Also Published As

Publication number Publication date
JP2001520051A (ja) 2001-10-30
AU1096799A (en) 1999-05-10
AU758541B2 (en) 2003-03-27
CA2308008A1 (fr) 1999-04-29
WO1999020779A1 (fr) 1999-04-29

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