EP1315798A2 - Cellules hotes pour empaqueter un virus adeno-associe recombine (raav), leur procede de preparation et leur utilisation - Google Patents

Cellules hotes pour empaqueter un virus adeno-associe recombine (raav), leur procede de preparation et leur utilisation

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Publication number
EP1315798A2
EP1315798A2 EP01967320A EP01967320A EP1315798A2 EP 1315798 A2 EP1315798 A2 EP 1315798A2 EP 01967320 A EP01967320 A EP 01967320A EP 01967320 A EP01967320 A EP 01967320A EP 1315798 A2 EP1315798 A2 EP 1315798A2
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European Patent Office
Prior art keywords
rep
aav
host cell
helper
cap
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EP01967320A
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German (de)
English (en)
Inventor
Joan Bertran
Ulrich Moebius
Markus HÖRER
Bernd Rehberger
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Medigene AG
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Medigene AG
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Publication of EP1315798A2 publication Critical patent/EP1315798A2/fr
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • rAAV recombinant adeno-associated virus
  • the present invention relates to a host cell for packaging recombinant adeno-associated virus (rAAV), the at least one copy of a first helper construct for the stable expression of at least one AAV Rep protein and at least one copy of another helper construct for the stable expression of at least one AAV cap -Proteins contains.
  • the invention further relates to helper constructs for the stable expression of at least one AAV Rep protein and AAV cap protein in a host cell, a vector construct which has one or more nucleic acids which are heterologous to AAV and methods for producing a host cell for packaging recombinant adeno-associated virus ( rAAV) and the use of the host cell for the production of rAAV.
  • the adeno-associated virus belongs to the parvovirus family. This virus family is characterized by an icosahedral, uncoated capsid with a diameter of approximately 18-30 nm, which contains a linear and single-stranded DNA of approximately 5 kb.
  • AAV adeno-associated virus
  • helper viruses for example with adenoviruses, herpes viruses or vaccinia viruses.
  • AAV inverted Terminal Repeats
  • ITR Inverted Terminal Repeats
  • rAAV particles recombinant AAV particles
  • Rep proteins non-structural viral proteins
  • Cap proteins structural viral proteins
  • a vector plasmid which carries the packaging genes flanked by the AAV ITRs carry transfected into cells suitable for packaging, eg HeLa or 293 cells, which are then infected with a suitable helper virus. After a few days, a lysate containing rAAV particles is obtained.
  • the capsid of the adeno-associated viruses naturally consists in a proportion of 1: 1: 10 from the three proteins VP1, VP2 and VP3.
  • the AAV capsid genes are located at the right end of the AAV genome and are encoded by overlapping sequences within the same open reading frame (ORF), using different start codons and a splice donor and two splice acceptor sites for their expression.
  • the VPl gene contains the entire VP2 gene sequence, which in turn contains the entire VP3 gene sequence with a specific N-terminal region. The fact that the overlapping reading frames code for all three AAV capsid proteins is responsible for the obligatory expression of all capsid proteins, albeit in different proportions.
  • AAV serotype 2 human AAV serotype 2
  • All serotypes represent virus vectors with advantageous properties for somatic gene therapy.
  • AAV vectors are therefore particularly well suited for use in gene therapy.
  • replication-deficient viruses have generally been developed which, although they infect a desired target cell and can transmit the nucleic acid encoded therein, to the cell, but no longer reproduce in this cell. This is achieved, for example, by deleting genes important for virus replication, such as the genes coding for the structural proteins, and, if appropriate, incorporating the foreign nucleic acid to be transmitted in their place.
  • the deleted genes must be made available as so-called helper genes in “trans” in order to compensate for the defect in a virus that is no longer capable of replication in the cell.
  • a suitable method for producing these large amounts of rAAV particles is the co-transfection of a eukaryotic cell with two recombinant AAV plasmids and subsequent infection with a helper virus (Chiorini JA et al. (1995) Human Gene Therapy, 6, 1531).
  • the second recombinant AAV plasmid contains the AAV genes which are required for the production of the particles (rep and cap genes).
  • the absence of the ITR regions in the helper construct is said to prevent the packaging of the rep and cap genes in the AAV particle and thus the formation of undesired wild-type AAN.
  • Suitable cells which are permissive, ie accessible, for both the recombinant AAN constructs and for a suitable helper virus are then transfected with the two AAN constructs.
  • the transfected cells production cells
  • adenovirus as a suitable Hei- fervirus
  • the expression of AAV genes the replication of the transferred foreign nucleic acid and the packaging and assembly of the recombinant AAV particles takes place.
  • the rAAV particles contain the foreign nucleic acid, flanked on both sides by the ITR regions, in the form of single-stranded DNA.
  • AAV as a general transduction vector for mammalian cells is given, for example, by Muzyczka N. (1992) Current Topics in Microbiology and Immunology, 158, 97.
  • rAAV particles A major disadvantage in the production of rAAV particles is the concomitant formation of replication-competent wild-type AAV (rcAAV) and the presence of helper viruses, for example adenovirus.
  • helper viruses for example adenovirus.
  • helper viruses for example adenoviruses as helper viruses lyse the infected cells and also cause cellular immune responses to adenoviral proteins.
  • adenoviruses are pathogenic for humans, they cause unspecific cold symptoms. Wild-type AAV can multiply in the presence of a helper virus and spread throughout the body.
  • rep and cap genes would be expressed under such conditions, which in turn would amplify rAAV genomes in the same cell and lead to new rAAV particles. This could spread the rAAV genomes throughout the body.
  • HeLa-based packaging cell lines were developed which had copies of the entire AAV genome without the flanking ITRs and in which the rep and cap genes were under the control of the native viral promoters.
  • the viral promoters P5, P19 and P40 are not active in the absence of helper virus infection (Inoue and Russell (1998) J. Virol. 72, 7024-7031; Gao et al. (1998) Human Gene Therapy 9, 2353-2362 ).
  • helper virus infection the expression of AAV genes is induced in these stably transfected HeLa cells.
  • the advantage of these cell lines is that rAAV particles can be formed on a large scale and reproducibly, which is particularly necessary for a commercial production process (Allen et al. (1997) J. Virol. 71, 6816-6822; Wang et al. (1998) J. Virol. 72, 5472-5480).
  • a disadvantage of using these cell lines is the fact that only a recombination event with the vector genome is required for the development of wild-type AAV, which is why such cell lines are not suitable for use in gene therapy due to the unmanageable risk.
  • the present invention is therefore based on the object to provide host cells in the form of packaging and production cells, as well as helper and vector constructs suitable for the production of rAAV, which allow the production of rAAV on a large scale, but thereby the formation of wild-type AAV in is essentially prevented.
  • a cap gene expression construct which, in addition to the promoter P40 which is absolutely necessary for the expression of the cap gene, additionally contains the regulatory sequences of the promoters P5 and P19.
  • fervirus is inducible.
  • Such a cap expression construct which had only the promoter region of P40, but not of P5 and P19, was not stably integrated in the host cells. This phenomenon is explained by the toxicity of the constitutively expressed cap protein for the host cell.
  • One object of the invention is therefore initially a host cell for packaging recombinant adeno-associated virus (rAAV) containing at least one copy of a first helper construct for expressing at least one AAV Rep protein and at least one copy of another helper construct for expressing at least one AAV cap protein ,
  • rAAV adeno-associated virus
  • the nucleic acids coding for the Rep protein and the Cap protein are functionally separated from one another and operatively linked to the natural regulatory sequences of AAV. These are in particular AAV's natural promoters.
  • the host cell additionally contains at least one copy of a vector construct.
  • a host cell is also referred to below as a production cell.
  • the host cell additionally contains at least one copy of a nucleic acid construct for at least one gene product of a helper virus and / or a cellular gene which is necessary for the production of rAAV.
  • the invention further includes a helper virus independent production cell of rAAV. In addition to the production cell, this contains the genes of a helper virus necessary for the production of rAAV and / or regulated cellular helper genes, so that these cells do not have to be infected with helper viruses for the production of rAAV.
  • nucleic acids coding for the Rep protein and the Cap protein are operatively linked to the three natural regulatory sequences of AAV, in particular to the natural promoters of AAV, means that large amounts of rAAV are obtained, but the Development of wild-type AAV is essentially prevented.
  • the adeno-associated virus is selected from the serotypes AAV1, AAV2, AAV3, AAV4, AAV5 and / or AAV6.
  • Capsid mutants of these serotypes are also included according to the invention.
  • capsid mutants are understood to mean that the AAV particles can contain a mutated capsid. This can include a mutation of one or more amino acids, one or more deletions and / or insertions.
  • Corresponding examples are known to the person skilled in the art from the following references: WO 99/67393, Grifinan M. et al. (2001) Mol Ther. 3 (6): 964-75, Wu P. et al.
  • protein and “polypeptide” are used synonymously in the context of the present invention and refer to a polymer of amino acids of any length. These terms also include proteins that have undergone post-translational modification steps, such as glycosylation, acetylation, or phosphorylation.
  • nucleic acid DNA
  • genes or “gene sequences” refer to a polynucleotide which has at least one open reading frame and which has the ability to form a certain protein by transcription and translation.
  • regulatory sequence is understood to mean a genomic region which regulates the transcription of a gene to which it is linked.
  • Transcriptionally regulatory sequences include at least one transcriptionally active promoter , but can also include one or more enhancers and / or terminators of the transcription.
  • operatively linked refers to the arrangement of two or more components. Because the components are related, they are allowed to perform their function in a coordinated manner.
  • a transcriptional regulatory sequence or promoter is operative with the coding sequence when the transcriptionally regulatory sequence or the promoter regulates or starts the transcription of the coding sequence.
  • a promoter operatively linked to a gene to be transcribed is generally referred to as a "cis" element to the coding sequence, but he is not necessarily in close proximity to the gene to be transcribed.
  • the terms “functionally independent units” or “functionally separate” mean that two or more genes do not overlap, the term “gene” also encompassing the corresponding promoter in addition to the coding sequence.
  • the two genes no longer overlap .
  • this is achieved by duplicating both parts of the coding sequence used together and the p40 promoter (see, for example, FIG. 1A).
  • This can mean different arrangements of the genes in a genome.
  • the genes can be located at different locations in the genome, be it integrated at different locations in the genome or localized on different plasmids or a mixture of these.
  • the genes can also be located side by side on the same DNA molecule, for example a chromosome or a plasmid, but each gene is controlled from its own promoter. Such an arrangement is likely, for example, when two genes are transfected together on different DNA molecules. These molecules can form concatems during transfection, which then integrate at one point in the genome, but still form functionally independent units.
  • telomere a genetic entity that is altered from that entity found naturally.
  • adeno-associated virus it means that the virus carries nucleic acid (s) which were produced by a combination of cloning, restriction and / or ligation steps and which do not naturally occur in the adeno-associated virus.
  • naturally promoter or “homologous promoter” as used in the context of the invention mean that the genetic unit of the promoter or the regulatory sequence comes from the same organism as the rest of the unit with which it is compared. Conversely, a “heterologous” or “non-natural promoter” means that the promoter has been separated from its natural coding sequence and has been operatively linked to another coding sequence.
  • the stable expression of a protein in a cell means that the DNA coding for the protein is integrated into the genome of the host cell and is therefore passed on to the daughter cells in a stable manner during cell division.
  • stable expression can mean that the DNA is episomal and is kept stable by an independent replication. This is achieved, for example, by known, in particular viral, replication systems consisting of an initiator protein (eg SV40 large T antigen, EBNA 1) and an origin of replication (eg SV40 ori, EBV oriP)
  • an initiator protein eg SV40 large T antigen, EBNA 1
  • an origin of replication eg SV40 ori, EBV oriP
  • helper constructs is understood to mean recombinant AAV plasmids which contain either the AAV rep genes and / or the AAV cap genes.
  • packet cell is understood to mean a host cell which contains one or more helper constructs but no vector construct.
  • the term “production cell” is understood to mean a host cell which contains both one or more helper constructs and one or more vector constructs.
  • This production cell can be dependent on the helper virus if it is infected with a helper virus in order to produce rAAV However, it can also be independent of helper virus if the cell carries the genes necessary for inducing rAAV production, for example under the control of one or more inducible promoters, and thus does not have to be infected with helper virus for the production of rAAV.
  • vector cell is understood to mean a host cell which contains one or more vector constructs but no helper construct.
  • the one helper constructs which are used for the production of the host cell according to the invention for packaging recombinant adeno-associated virus contain nucleic acid sequences coding for at least one Rep protein, the proteins Rep 78, Rep 68, Rep 52 and Rep 40, especially Rep 68, Rep 52 and Rep 40, especially Rep 68 and Rep52 can be understood.
  • the other helper constructs contain nucleic acid sequences which code for at least one of the known cap proteins, the cap proteins being the proteins VP1, VP2 and VP3.
  • the genes for these proteins and the ITR sequences can be isolated from wild-type AAV, which are generally available in the form of clones. For example, the clone pSM620 in Samulski, et al. (1982) Proc.
  • helper constructs for expressing the Rep protein and the Cap protein are integrated into the genome of the host cell as functionally independent units. This effectively prevents the formation of wild-type AAN (rcAAV), because two recombination events would be necessary for the formation of wild-type viruses, which are quite rare with a frequency of 10 "7 per cell division, ie a total of 10 " 14 . In fact, no rcAAV could be detected in a recombinant virus preparation containing 2x 10 10 genomic particles.
  • the expression of the Rep protein by the natural AAV promoter P5 and the expression of the cap protein by the natural AAV promoter P40 in particular by the natural AAV promoters P19 and P40, especially by the natural AAV promoters P5 , P19 and P40 controlled.
  • the cap expression plasmid contains the AAV promoters P5, P19 and P40 in order to allow regulated expression depending on both helper virus infection or helper virus gene products and on rep protein expression, since this arrangement best reflects the natural lytic AAV life cycle. This arrangement proved to be very suitable for strictly regulated cap protein expression.
  • the expression of the Rep protein and the Cap protein in the host cell are regulated dependent on one another.
  • This approach was chosen because it was found that for efficient packaging of rAAV in stable cell lines, weak cap expression is required first, since otherwise high amounts of cap have a toxic effect on the cells.
  • strong cap expression must occur at the time of packaging.
  • a constitutive, heterologous promoter cannot meet both of these criteria at the same time. Although this can be improved by using inducible, heterologous promoters, the exact time regulation and the level of cap expression by such promoters are extremely difficult to implement in practice.
  • the expression of Cap is coupled to the activation by helper virus gene products and / or cellular helper genes and Rep and is therefore controlled in time exactly as in the wild-type situation.
  • the transcription of the nucleic acids coding for the Rep proteins and the cap proteins is particularly advantageously terminated by the natural regulatory sequences, in particular by the natural AAV poly-A signal. Similar to the initiation of transcription, the use of the homologous sequences to terminate the transcription of the AAV cap and rep genes increases the amount of rAAV particles produced by the AAV vector system.
  • a mammalian cell in particular a human cervical carcinoma cell, in particular a HeLa cell, is suitably used as the host cell.
  • HeLa cells have proven to be particularly advantageous because the AAV P5 promoter in HeLa cells is almost inactive and it is therefore possible to stably integrate an expression cassette for the AAV Rep protein into their genome under the control of the natural regulatory elements, so that the Rep protein is not toxic in these cells (Clarke et al. (1995) Human Gene Therapy 6, 1229-1341; Tamayose et al. (1995) Human Gene Therapy 7, 507-513; Inoue & Russell (1998 ) supra, Gao et al. (1998) supra).
  • first helper construct for the stable expression of at least one AAV Rep protein in a host cell
  • the nucleic acid coding for the Rep protein being operable with the natural regulatory sequences of AAV, in particular with the natural AAV promoters P5 and P19 are connected
  • a second helper construct for the stable expression of at least one AAV cap protein in a host cell, the nucleic acid coding for the cap protein being operatively linked to the natural regulatory sequences of AAV, preferably using the natural AAV promoter P40 , in particular with the natural AAV promoters P19 and P40, especially with the natural AAV promoters P5, P19 and P40.
  • An advantage of the separation of rep and cap into different expression units is that by replacing the cap gene or the cap expression unit with a cap gene or a cap expression unit of another AAV serotype, an AAV particle of another serotype is generated can be used, wherein the same rep gene or the same rep expression unit and the same vector construct can be used. This therefore minimizes the effort if, for various reasons, different AAV serotypes are to be used for the same vector construct.
  • Another object of the present invention is a helper construct containing nucleic acid sequences coding for at least one Rep protein, the Rep proteins being Rep 68, Rep 52 and / or Rep 40, but not Rep 78, because it was surprisingly found that in addition to Rep 52, Rep 40 and the three cap proteins VP1, VP2 and VP3 the additional expression of only Rep 68 is sufficient for packaging AAV vectors.
  • the advantage of these Rep 78 deficient helper constructs is that the largest Rep protein, which is most toxic to the packaging cells, is not expressed at all. It was also found that Rep 78 has the greatest inhibitory activity on cellular processes, such as transcription, among the Rep proteins. Therefore, when using this helper construct, the packaging efficiency can be increased due to the absence of Rep 78.
  • Rep 68 and Rep 78 are expressed in the natural system by the p5 promoter.
  • the use of the Rep 78-deficient helper construct is also advantageous because, compared to Rep 78 in Adeno virus-infected cells, Rep 68 is the stronger transactivator of the AAV promoters P19 and P40 (Hörer et al. (1995) J. Virol. 69, 5485 -5496; Weger et al. (1997) J. Virol. 71, 8437-8447). Therefore, the use of this Rep 18-deficient helper construct leads to an increased expression of the smaller Rep proteins Rep 40 and Rep 52 as well as the capsid proteins and thus the desired higher packaging efficiency.
  • the AAV sequences from nucleotide 201 to nucleotide 4497 including the deletion of the intron sequence and from nucleotide 658 to nucleotide 4460 were used cloned into the bacterial expression plasmid pUC19, the binding sites for the Rep protein in the pUC19 sequence being deleted (cf. FIG. 6).
  • two rep and at least two cap genes, each with its own poly (A) sequence are arranged one behind the other for the termination of the transcription.
  • Rep proteins Rep 68 and Rep 40 and cap proteins VP2 and VP3 can be expressed from the first section (AAV sequence nucleotide 201 to nucleotide 4497), while starting from the second section (AAV sequence nucleotide 658 to nucleotide 4460) the Rep proteins Rep 52 and Rep 40 and the cap proteins VP1 VP2 and VP3 are expressed. All AAV-2 proteins with the exception of Rep 78 are encoded.
  • the AAV sequences mentioned (nt 201-2310; nt 658-4460 including the deletion of the intron sequence) were also incorporated into the bacterial expression plasmid pUC19 cloned (see FIG. 6).
  • the binding sites for the Rep protein in the pUC19 sequence were again deleted. In this way, the rep gene was partially duplicated.
  • the resulting helper construct contains only one poly (A) sequence, so that all mRNA transcripts have the same 3 'end.
  • the Rep proteins Rep 68 and Rep 40 can be expressed starting from the first section (AAV sequence nucleotide 201 to nucleotide 2310), while starting from the second section (AAV sequence nucleotide 658 to nucleotide 4460) the Rep proteins Rep 52 and Rep 40 and the cap proteins VP1, VP2 and VP3 are expressed. All in all, this vector construct also encodes all AAV-2 proteins with the exception of Rep 78.
  • helper construct pUC " ⁇ Rep78 ⁇ Cap” (RBS) ⁇ 37 for expression of the Rep proteins Rep68, Rep52 and Rep40, the AAV nucleotides 2945 to 4046 from the cap gene (nucleotides 2203 to 4410) of the helper construct pUC " ⁇ Rep78Cap” (RBS ) Deleted ⁇ 37. This deletion means that functional cap proteins can no longer be expressed.
  • the present invention furthermore relates to vector constructs containing one or more nucleic acids which are heterologous to AAV and which are derived from ITR Sequences are flanked, the 5'-localized ITR sequence having a deletion in the region of the C-palindrome.
  • vector constructs contain the AAV sequences 1-60 / 83-191 ( ⁇ C arm ITR as left ITR - explanation in the following) and 4498 to 4671 (as right ITR).
  • the AAV-2 ITR sequences which are 145 base pairs long, are composed of one large palindrome (A) and two smaller palindromes (B and C).
  • the first 125 bases of the ITR sequence form a hairpin structure in a T-shape (see e.g. Muzyczka, N. (1992), supra).
  • the terminal sequence can be in one of two configurations. In the first configuration, also called “flip”, the B palindrome is closer to the 3 'end and in the second configuration, also called "flop", the C palindrome is closer to the 3' end (see also Svivstava, A. et al. (1983) J. Virol. 45 (2), 555).
  • the two configurations frequently change their configuration due to a recombination event, which destabilizes the corresponding vector constructs.
  • a deletion within the 5'-flanking ITR sequence which in a preferred embodiment comprises 80 nucleotides, in particular 40 nucleotides, especially 22 nucleotides in the range from nucleotide 61 to 82, brings about stabilization of these vector constructs, because a change of configuration between flip-flop orientations is no longer possible (see FIG. 8).
  • vector constructs which have such a deletion in the C-palindrome of the 5'-ITR sequence can be packaged just as efficiently as vector constructs with intact ITR sequences.
  • Another object of the present invention is therefore a vector construct containing one or more nucleic acids which are heterologous to AAV and are flanked by ITR sequences, the 5'-localized ITR sequence having a deletion in the region of the C-palindrome.
  • the present invention furthermore relates to a vector cell containing a vector construct, the 5 '-located ITR sequence having a deletion in the region of the C-palindrome.
  • Another object of the present invention relates to a vector construct containing one or more nucleic acids heterologous to AAV, in particular a nucleic acid coding for a protein selected from a cytokine, in particular IL2, IL4, IL12 and / or GM-CSF (granulocyte-macrophage-colony-stimulating) Factor) and / or a co-stimulating molecule, in particular B7, especially B7.1 and / or B7.2.
  • a cytokine in particular IL2, IL4, IL12 and / or GM-CSF (granulocyte-macrophage-colony-stimulating) Factor
  • B7 especially B7.1 and / or B7.2
  • any coding or non-coding nucleic acid sequence can be used as the heterologous nucleic acid sequence.
  • one or more heterologous nucleic acid sequence (s) are introduced into a replication-deficient vector construct by conventional cloning techniques known to those skilled in the art (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • nucleic acid sequences are coding sequences for chemokines such as lymphotactin, Rantes, MCP-1 or Mip l ⁇ , cytokines such as IL12, IL7, IL18, IL2, GM-CSF, IL1, IL6, interferon ⁇ or IL-10, or antibodies, Antibody fragments or single-stranded antibodies, for example directed against ICOS, also against the ICOS receptor, CD40, CD40 ligands, VEGF, IL-1, TNF, against tumor antigens such as Her-2 / neu, GD3 or CA125, against viral antigens or against IgE; furthermore against soluble receptor forms such as ICOS FC, ICOS ligand FC, CD40L FC, TNF- ⁇ receptor FC, against apoptosis inducing molecules such as proteins of the BCL-X family, BAX, BAD or caspases, necrosis-inducing peptides such as perforins, toxins, for example
  • non-coding sequences are suitable for use as ribozymes or antisense RNAs.
  • a genetically modified melanoma cell line which expresses GM-CSF can be used as a vaccine.
  • WO 94/16716 discloses the use of a recombinant virus in cancer therapy using at least one cytokine, for example GM-CSF or B7 and / or a tumor-associated antigen.
  • the B7 gene here refers to the so-called B7.1 gene.
  • WO 94/04196 discloses a DNA construct for the treatment of tumor diseases which codes for a cytokine and also for B7, here also B7.1.
  • WO 92/00092 is a nucleic acid sequence coding for B7.1
  • WO 94/03408 and WO 95/06738 is a nucleic acid sequence coding for B7.2 and from EP-B1-0 188 479 a nucleic acid sequence is coding for GM-CSF known.
  • B7.2 is particularly advantageous because in vitro studies have shown that, in contrast to interferon ⁇ or IL12, the B7.2 protein in connection with B7.1 has an inhibitory effect on the activation of T Lymphocytes (Rudy et al. (1997) Int. Immunol., 9, 853).
  • the tumor-destroying effect of a B7 molecule can be increased.
  • Another object of the present invention is therefore the aforementioned vector construct containing one or more nucleic acids heterologous to AAV, in particular a nucleic acid coding for a protein selected from a cytokine, in particular IL2, IL4, IL12 and / or GM-CSF and / or one co-stimulating molecule, in particular B7, especially B7.1 and / or B7.2.
  • a cytokine in particular IL2, IL4, IL12 and / or GM-CSF and / or one co-stimulating molecule
  • B7 especially B7.1 and / or B7.2.
  • so-called double vectors which contain both the nucleic acid sequence coding for GM-CSF and the nucleic acid sequence coding for B7 are particularly preferred (cf. FIG. 7).
  • the use of double vectors in particular reduces the number of packaging processes.
  • the double vectors according to the invention allow a comparable efficient expression of both foreign nucleic acids as the co-infection with two single vectors (cf. also FIG. 7).
  • the heterologous nucleic acids are flanked by AAV ITR sequences and their expression is regulated by a promoter and / or enhancer heterologous to AAV, in particular by the major immediate early enhancer / promoter (MIEP) of the cytomegalovirus (CMN).
  • MIEP major immediate early enhancer / promoter
  • CMN cytomegalovirus
  • Suitable promoters are all promoters which are heterologous to AAN and which are active in eukaryotic cells, preferably mammalian cells. These are, for example, the SN40 promoter (Samulski et al. (1989) J. Nirol.
  • the CMV-MIEP is particularly preferred due to its very strong expression, which is subject to only the slightest fluctuations.
  • Another object of the present invention is a process for the production of a host cell for packaging and / or production of recombinant adeno-associated virus (rAAV), which comprises the steps:
  • Rep helper construct and the Cap helper construct can also be reversed.
  • a Rep helper construct and a cap helper construct can be introduced essentially simultaneously.
  • helper virus-independent packaging cell can be produced from rAAV, comprising the additional steps
  • helper gene of the helper viruses and / or a regulated cellular helper gene into the production cell (f / e) selection of a vector cell containing the helper gene (s).
  • helper genes are understood to mean the genes of the AAV helper viruses and / or cellular genes whose gene products are necessary for the replication of the AAV or promote it.
  • the helper genes are E1A, E1B, E4, E2A and VA.
  • El A is required for the transactivation of the AAV p5 promoter.
  • the gene products E1B and E4 serve to increase the AAV mRNA accumulation.
  • the gene products E2A and VA serve to enhance AAV mRNA splicing and translation.
  • Herpes simplex virus (HSV) helper genes are also included according to the invention as helper genes. According to a preferred embodiment, these are the 7 replication genes UL5, UL8, UL9, UL29, UL30, UL42 and UL52. UL 5, 8 and 52 form the HSV helicase-primase complex, UL29 codes for the single-stranded DNA binding protein, UL42 for a double-stranded DNA binding protein, UL30 codes for the Finally, HSV DNA polymerase and UL9 codes for a protein which binds the HSV origin of replication (see Weindler FW and Heilbronn R (1991) J Virol. 65. (5): 2476-83).
  • HSV Herpes simplex virus
  • helper virus instead of the individual helper genes, for example the adenovirus type 5 (Ad5), is particularly advantageous because this comes as close as possible to the natural situation of AAV multiplication in the presence of helper viruses and thus the packaging of rAAV particles very efficiently is.
  • helper viruses are, for example, herpes viruses or vaccinia viruses.
  • a component of the invention is the production of a vector cell from rAAV, comprising the steps:
  • helper virus independent vector cell of rAAV can be produced, comprising the additional steps
  • helper virus-independent vector cell correspond to those of the helper virus-independent packaging cell.
  • the present invention further relates to a method for producing a production cell of rAAV, which comprises the steps:
  • Another way to manufacture a production cell consists of the steps:
  • Another object of the invention relates to the production of a helper virus-independent production cell of rAAV, which comprises the additional steps:
  • introducing constructs is generally understood to mean transfection.
  • the construct cannot be permanently integrated into the genome of the host cell, which is generally referred to as transient transfection.
  • the construct, in particular the vector construct can also be stably integrated into the genome of the host cell or can be retained as a stable episomal copy (using a replication system, for example from SV40 large T antigen / SV40 ori or EBNA 1 / EBV oriP) ,
  • a replication system for example from SV40 large T antigen / SV40 ori or EBNA 1 / EBV oriP
  • the construct (s), in particular the vector construct (s), are introduced by infection with viruses.
  • Recombinant viruses are preferred here, where, for example, rAAV, adenoviruses, herpes viruses, vaccinia viruses, baculoviruses and / or phages, in particular bacteriophages, can be used.
  • a preselection for cells with integration events is appropriate for the above-mentioned methods for producing packaging, vector and / or production cells for the selection of cells with stably integrated constructs.
  • a 10: 1 reporter construct can be added to the Rep or Cap helper construct to be transfected and introduced together, for example co-transfected, into the host cell.
  • a selection is then made with regard to the reporter, since it can be assumed that the respective helper construct has also been integrated with high probability into cells in which the reporter construct has been integrated.
  • the corresponding cells can be selected in each case by detecting the protein expression, for example using a Western emblot.
  • the quantitative detection of a construct-specific nucleic acid in the cells would also be suitable, for example via a quantitative polymerase chain reaction (PCR), a Southern or Northern blot.
  • PCR quantitative polymerase chain reaction
  • a preferred form of detection for suitable cells is also the direct detection of the packaging of rAAV in these cells, in that missing constructs for the packaging are introduced into the respective cell and the packaging is initiated by infection with a helper virus.
  • the other helper construct and a vector construct for example with a color marker such as GFP as the transgene, are required.
  • a vector construct is required for a packaging cell with Rep and Cap helper constructs.
  • Rep and cap helper constructs are required for a vector cell.
  • a helper nor a vector construct is necessary for a production cell.
  • These cells can then be introduced by introducing the viral helper genes, in particular especially induced by infection with a helper virus for rAAV production.
  • the helper virus-independent packaging, vector or production cells do not require the introduction of helper genes, provided that they already contain all the necessary helper genes or only the introduction of individual helper genes. For these cells, only the helper genes need to be induced to start rAAV production.
  • the AAV titer can then be determined using customary methods, which then serves as a measure for optimal cap or cap / rep expression.
  • This procedure has the advantage that the selection of the corresponding cells not only selects for absolute expression amounts, but also for the optimal ratio of cap to rep expression.
  • this method has the advantage that a selection for intact Rep and Cap genes is also carried out here, since mutants of Rep and / or Cap which still form protein, but which are no longer suitable for packaging rAAV, are used in the other mentioned detection methods would not be recognized.
  • Another object of the invention relates to the use of a Rep helper construct and / or a cap helper construct and / or a vector construct, or a packaging cell, a vector cell or a production cell, in particular a Herfer virus-independent production cell for the production of rAAV.
  • the rAAV vector constructs contain one or more nucleic acids heterologous to AAV.
  • GM-CSF and B7.1 and / or B7.2 are used as the heterologous nucleic acid, as already described above
  • the rAAV particle resulting from the packaging process which carries the two immunostimulatory genes, can be used as efficient transduction vector for the treatment of various tumor diseases, for example melanoma or ovarian cancer, can be used.
  • the figures and the examples below are intended to explain the invention in more detail, but without restricting it.
  • Figure 1A shows a schematic representation of some helper constructs according to the invention in comparison to wild-type AAV.
  • the natural AAV promoters P5, P19 and P40 are shown as well as the "major intron” of the AAV genome (“I”) and the natural poly (A) signal of the AAV genome (“pA”) coding sequences for the rep and cap genes of AAV shown.
  • FIG. 1B shows a schematic representation of three different vector constructs, the ITRs being shown at the left and right flanking ends.
  • CMV stands for the "major early promoter” of the cytomegalovirus.
  • I indicates an intron which is contained in the plasmid pCI from Promega.
  • pA indicates the poly (A) signal of the Simian Virus 40 (SV40).
  • GFP green fluorescent protein
  • B7.2 for the immuno-stimulatory protein B7.2
  • GM-CSF for granulocyte / macrophage colony stimulating factor
  • NLacZ stands for the nuclear localized form of the enzyme ß-galactosidase.
  • FIG. 2 shows the identification of the stable Rep cell lines by means of a Western blot experiment.
  • HeLa cells were transfected with the Rep helper construct (P5 Rep), the Rep expression being controlled by the natural AAV promoters P5 and P19.
  • the selection marker hygromycin which was transfected 1:19 to the Rep helper construct shown, is not shown.
  • several hygromycin-resistant cell clones were picked and the Rep expression with adenovirus induced as helper virus (lane 1-8).
  • the total amount of cellular protein was obtained and analyzed by Western blot with using a specific Rep antiserum.
  • the numbers 78/68/52/40 refer to the Rep proteins Rep 78, Rep 68, Rep 52 and Rep 40.
  • "M” stands for the molecular weight standard, "+" for positive control and "-" for negative control.
  • FIG. 3 shows the inducible cap gene expression starting from various constructs.
  • the cap helper constructs shown in the lower area were used to transfect the Rep expressing cell line R84. Subsequently, the cells were not infected with adenovirus (MOI5) (lanes 1 to 4 and wt marked with "+”) or as a control (lanes 1 to 4 and wt marked with "-"). After 72 hours, the total cellular protein content was examined by Western blot analysis for the expression of the three cap proteins VP1, VP2 and VP3.
  • MOI5 adenovirus
  • FIG. 4 shows the inducible expression of the Rep and Cap proteins in the stable packaging cell line C97 after adenovirus infection.
  • C97 cells or HeLa control cells were either infected with adenovirus (MOI5) or not infected as a control.
  • Whole cell protein was extracted 72 hours after infection and analyzed for its presence by Western blot using antibodies specific for the Cap and Rep proteins.
  • the four Rep proteins, Rep 78, Rep 68, Rep 52 and Rep 40, the cap proteins VP1 and VP2 and, to a greater extent, the cap protein VP3 can be clearly recognized in the packaging cell line C97 ,
  • FIG. 5 shows the replication of recombinant AAV (rAAV) in C97 cells, the rAAV replication intermediates being detected as low molecular weight DNA isolated from C97 cells which had previously been infected with rAAV and adenovirus (MOI5).
  • rAAV recombinant AAV
  • MOI5 adenovirus
  • the supernatant was then used to infect fresh C97 cells with new helper viruses (MOI5).
  • MOI5 new helper viruses
  • a clear cytopathic effect was again observed, whereby DNA with a low molecular weight was isolated (second round of amplification).
  • the isolated DNA from the first and second rounds of amplification was analyzed by Southern blot, the vector construct according to the invention being used as a probe for the presence of the replication intermediates of the AAV genomes in order to obtain an infectious titer of an original AAV stock.
  • FIG. 6 schematically shows further helper constructs, all of which are derived from the cloning vector pUC19.
  • Figure 7 shows schematically single and double expression vectors.
  • FIG. 8 schematically shows the 5 '-located ITR sequence of the AAV vector constructs with the configurations flip, flop and deletion of the C-palindrome, which is designated as " ⁇ (C-arm)".
  • FIG. 9 schematically shows the nucleotide sequence of the 5 ′ -located ITR sequence of the different configurations from FIG. 8.
  • FIG. 10 shows the nucleotide sequence of the 5 '-located ITR sequence with and without deletion and of the 3' -located ITR sequence.
  • FIG. 11 schematically shows the equally efficient packaging of a vector construct with a deleted 5 '-located ITR sequence [ ⁇ AAV- (B7.2free + GM-CSF)] compared to a vector construct with a 5' -located ITR sequence in a flop orientation Configuration [( ⁇ AAV- (B7.2 + GM-CSF)].
  • FIG. 12 shows schematically a Rep 78-deficient helper plasmid with the designation pUC "Rep68,52,40Cap” (RBS) ⁇ 37.
  • FIG. 13 schematically shows a further Rep 78-deficient helper plasmid with the designation pUC " ⁇ Rep78Cap” (RBS) ⁇ 37.
  • Plasmid P5 Rep (Fig. 1A) was prepared by deleting a DNA fragment containing nucleotides 2300-4170 of the AAV genome. (Ruffing et al. (1994) J. Gen. Virol. 75, 3385-3392 (Gene Bank Accession No. AF 043303). P5 Rep ⁇ 37 was obtained from P5 Rep by deleting the AAV bases 4461-4497.
  • Plasmid P5P19P40Cap ( 1A) was obtained by deleting the DNA segment between nucleotides 350 to 650 and 1045 to 1700.
  • P5P19P40Cap ⁇ 37 was obtained from P5P19P40Cap by deleting AAV bases 4461-4497.
  • Plasmids 1-4, which are shown in FIG contain various deletions of the AAV genome. Plasmid 1 lacks the sequence between the Bell and Hindlll cleavage sites of the AAV2
  • Plasmid 2 lacks the sequence between the Nrul and BstEII cleavage sites
  • plasmid 3 lacks the sequence between the Ba HI and BstEII cleavage sites.
  • Plasmid 4 corresponds to plasmid P5P19P40Cap from FIG. 1A.
  • the vector constructs for rAAV-B7.2-GM-CSF and rAAV-GFP were constructed using the pCI plasmid from Promega (Germany) and then converted into a pUC19-based plasmid which had the ITR sequences (cf. PCT / EP00 / 01090).
  • the vector construct nLacZ which is also used as a control, has already been described in the literature (Bertran et al. (1996) J. Virol. 70, 6759-6766). Packaging of various vector constructs
  • AAV bases 190 to 1060 were amplified from wild-type AAV DNA by means of PCR.
  • the 5 'primer was chosen so that a unique Xbal interface was introduced at position 199.
  • the PCR fragment was cut with Xbal and BamHI and cloned into the similarly cut vector pUC19.
  • Position 199 in the AAV genome was chosen to ensure that all important P5 promoter elements are present in the helper construct.
  • wild-type AAV DNA was cut with BamHI and SnaBI and the insert fragment was then cloned into the BamHI and Smal position of the intermediate. In this way, the basic helper construct pUC "rep / cap" was obtained (FIG. 6).
  • This helper construct contains the AAV sequences 201 to 4497, while the vector construct carries the AAV sequences 1 to 191 or 1-60 / 83-191 (left ITR) and 4498 to 4671 (right ITR) (see FIG. 7) , In this way it was ensured that there were no homologous AAV sequence overlaps on the plasmids. ⁇ 37 bases were then deleted from the 3 ′ end of the AAV genome in pUC “rep / cap”, which bases are not required for optimal expression of the AAV Rep and cap genes. The resulting helper construct is designated pUC "rep / cap” ⁇ 37 and contains the minimalized AAV sequence from positions 201 to 4460 (FIG. 6).
  • the Rep binding site (RBS) in the vector backbone pUC19 was then deleted in order to avoid non-homologous recombination at this position.
  • the construct is called pUC "rep / cap” (RBS) ⁇ 37 (see FIG. 6).
  • the three RBS in the Rep gene (P5 and P19 promoter) were also mutated plasmids individually and in various combinations without changing the amino acid sequences of the Rep protein (FIG. 6).
  • a so-called “functional separation” sequence (fs) of 638 base pairs in length was introduced between the Rep and Cap genes.
  • the AAV sequence 1691 to 2328 which contains the Rep C terminus or the AAV P40 promoter including cap sequences and all the regulatory sequences essential for P40, was doubled and placed behind the stop codon for the spliced Rep - Versions (heading 2329) Complained.
  • the P40 promoter in the rep gene was subsequently destroyed without changing the Rep amino acid sequence. This was ensured by mutating the P40 TATA box.
  • the further Rep 78-deficient helper constructs were produced by deleting the AAV nucleotides 1907 to 2227, which correspond to the rep intron, in the helper construct pUC "rep / cap” (RBS) ⁇ 37 (cf. FIG. 6) by double-strand mutagenesis , whereby the plasmid pU-
  • CAAV splice was obtained as an intermediate.
  • the plasmid pU-CAAN splice was linearized with the restriction enzyme ,del, treated with an exonuclease, for example mung bean ⁇ uclease (Boehringer Mannheim, Germany) and then mixed with the restriction enzyme SphI.
  • the thus obtained fragment with a length of 4222 bp was connected with a nector fragment to the Rep78-deficient helper construct pUC "Rep68,52,40Cap” (RBS) ⁇ 37 (10657 bp) by ligation (cf. FIG. 12).
  • pUC "rep / cap” (RBS) ⁇ 37 (cf. FIG. 6) can be used to obtain this nector fragment, which is obtained after treatment with the restriction enzymes Nrul and SphI in a length of 6435 bp.
  • the same nector fragment can also be used to produce another Rep78-deficient helper construct ⁇ UC " ⁇ Rep78Cap” (RBS) ⁇ 37 (see FIG. 13).
  • the intermediate pU-CAANSpl substrate was treated with the restriction enzymes Asel, BsrBI and SphI.
  • the 1808 bp BsrBI-Sphl fragment was digested with the 6435 bp nector fragment for said helper construct pUC " ⁇ Rep78Cap” (RBS) ⁇ 37 with a total length of 8243 bp
  • Rep78-deficient helper constructs show, in sequential transfection of helper and vector construct (experiments 1 and 3, shown in table 4 below, and a further experiment in table 5), approximately equally good packaging efficiencies compared to rep78-encoding helper constructs.
  • experiment 2 co-transfections were carried out, which means that different experimental conditions were selected, and therefore experiment 2 cannot be compared with experiments 1 and 3 in terms of packaging efficiency.
  • Table 4
  • Rep helper constructs p5Rep ⁇ 37 encodes all 4 Rep proteins, but no cap) or ⁇ UC “ ⁇ Rep78 ⁇ Cap” (RBS) ⁇ 37 (codes exclusively for the Rep proteins Rep68, 52 and 40) together with the cap construct p5pl9p40Cap ⁇ 37 in a ratio of 1 : 1 co-transfected into suitable packaging cells
  • subsequent sequential or simultaneous transfection of a vector construct and overinfection with adenoviruses can in turn achieve comparable rAAV titers, as if a single helper construct which codes for both rep and cap are transfected into the cells ( pUC "rep / cap” (RBS) ⁇ 37, pUC "Rep68,52,40Cap” (RBS) ⁇ 37, pUC " ⁇ Rep78Cap” (RBS) ⁇ 37).
  • Table 5 Surprisingly, no rcAAV impurities have so far been found in rAAV lysates
  • the cells Two days after (the first) transfection, the cells were infected with Ad-5 (MOI2). Three days later, the cells in the medium were disrupted by freeze / thaw lysis, cell components were pelleted and the rAAV lysate was heat-inactivated at 60 ° C. for 10 min. 3 ⁇ 10 5 irradiated HeLa-t cells (100 Gy) were infected with various dilutions of the lysate. 40 hours after infection of the cells with rAAV, the cells were analyzed in the FACS flow (see below) for GFP expression and the transducing titers of the rAAV crude lysates were determined from this. Each helper construct was tested in at least five independent experiments.
  • Vector plasmid pAAV- (B7.2free / GM-CSF) tested. This was examined on the one hand in a triple transfection (transfection of all three plasmids simultaneously), on the other hand in a sequential transfection, in which the two AAV helper plasmids and - one day later - the vector plasmid were transfected.
  • helper plasmids according to the invention, both higher packaging efficiencies were achieved and the formation of rcAAV was prevented.
  • the HeLa parent cells and all packaging cell lines derived therefrom were kept as "monolayer" cell cultures in Dulbecco's Modified Eagle's Medium (DMEM), which had 10% fetal calf serum.
  • various selection agents were used in order to stabilize the transfected cells
  • hygromycin B in a concentration of 500 ⁇ g / ml
  • neomycin in a concentration of 800 ⁇ g / ml
  • puromycin in a concentration of 1 ⁇ g / ml were used C. in a 5% CO atmosphere.
  • the transfections were carried out using conventional calcium phosphate precipitation techniques (Ca 3 (PO 4 ) 2 ) and endotoxin-free plasmid DNA, which were carried out using kits from Company Qiagen (Hilden, Germany), for stable
  • a Rep-coding plasmid was produced by removing the cap-coding sequences from the genome of AAV2, which had no ITR sequences but all other AAV regulatory elements (FIG. 1). HeLa cells were then transfected with the Rep expression construct, and cell clones which replicated the Rep protein
  • plasmids were constructed in which the expression of the cap protein was controlled by the natural AAV regulatory elements.
  • a plasmid was constructed that had the cap gene under the control of the P5 promoter. This plasmid resulted in detectable expression of cap protein only after helper virus infection with adenovirus, but the detectable levels of protein were comparatively low.
  • a second plasmid was therefore constructed in which the DNA sequences between the P5 and the P19 promoter were removed. This plasmid not only expressed the Cap proteins, but also the P19 Rep protein. With the help of this cap expression After helper virus infection, plasmids could only be detected in a very small amount of cap protein.
  • cap plasmids were then constructed, which are shown schematically in FIG. 3. All of these plasmids led to a significant cap protein expression, the protein amounts obtained being comparable to the protein amount of a plasmid which contained the entire AAV genome with the exception of the ITR sequences (cf. FIG. 3 "wt").
  • the plasmid P5P19P40Cap as shown in FIG. 1, was used for all subsequent experiments because, in a wide variety of experiments, it always delivered slightly higher amounts of cap protein than the other cap proteins.
  • the cell line stably expressing the Rep protein described in Example 5 was then transfected with the plasmid P5P19P40Cap. Subsequently, a cell clone was identified, which according to adenovirus
  • a decisive advantage of the cell line according to the invention is that no wild-type AAV (rcAAV) is to be expected. Numerous attempts have been made to detect rcAAV contamination, but no rcAAV particles have so far been detected in virus stocks made from these cell lines. 1-2 ml of virus were tested with about 5 x 10 7 transducing particles / ml.
  • a next step stable cell lines for the production of rAAV should be produced, which no longer require transfection of the vector construct for the rAAV production and thus allow a large-scale production of rAAV.
  • several independent Common transfection steps transfected vector constructs in C97 cells.
  • clones were screened for the expression of the foreign DNA contained in the rAAV genomes.
  • a selection between the clones was also made with regard to the virus yield by means of rep and cap expression.
  • helper plasmids p5Rep ⁇ 37 (coding for all 4 Rep proteins) and pUC " ⁇ Rep78 ⁇ Cap” (RBS) ⁇ 37 (Rep78-deficient) in combination with p5pl9p40Cap ⁇ 37 (coding for the 3 capsid proteins) and a neomycin resistance gene plasmid were described as follows Packaging cell lines made for AAV-2 vectors.
  • HeLa-t cells were co-transfected at approx. 10% confluence with a rep, cap and resistance gene plasmid (in a ratio of 10: 10: 1) and for as long cultivated further until the cells reached confluency of almost 100%.
  • Antibiotic selection G418 was started 48h after the transfection. After reaching 100% cell confluence, the cells were trypsinized and seeded in 10 cell pools, each with 10% confluence. These cell pools were further cultivated for about 4 weeks (with each passenger - cell formation after reaching 100% confluence) before a first packaging experiment was started.
  • Replicas were made from each pool. A replica was cultivated further, the associated replica was at 10% confluence with a
  • packaging cells were transfected with a vector plasmid and infected 48 hours later with adenovirus (MOI 5).
  • Production cell lines were only infected with adenovirus.
  • the cells and the supernatant were harvested 72 hours after the adenovirus infection, the cells being centrifuged off by centrifugation at 1500 rpm for 5 min and approximately 10 7 cells / ml being resuspended in part of the supernatant.
  • the remaining part of the supernatant was treated at 56 ° C for 30 min and then frozen until use.
  • the cell suspension was subjected to a three "freeze-thaw" cycle, the freezing process being carried out at -80 ° C. and the thawing process at 37 ° C. Subsequently, cell debris was removed
  • HeLa cells were exposed to UV light (35 J / m 2 ) in a Stratalinker TM (commonly available from the
  • virus stocks used in the context of this invention were titrated using a special infection test, with two rounds of amplification of the packaging cells being carried out in order to determine the infectious titer of the preparations. Details of this experiment can be found, for example, in Clark et al. (1996) Gene Therapy 3, 1124-1232.
  • the virus stocks were serially diluted in serum-free medium, and then portions containing known volumes of the starting virus stocks were used to transduce the packaging cell line C97 in the context of this invention together with a constant amount of adenovirus (MOI5). After 72 hours the cells and the supernatant were harvested, subjected to a three "freeze-thaw” cycle, centrifuged to remove cell parts, treated at 56 ° C.
  • DNA encapsulated was encapsulated by subsequent incubation with ProteinaseK (0.5 mg / ml) in 0.5% SDS released.
  • the DNA was then purified via a phenol / chloroform extraction, then subjected to ethanol precipitation and blotted onto a nylon membrane using a dot blot apparatus, and then hybridized with probes that were specific for rAAV or wild-type AAV.
  • the ratio between genomic particles and infectious particles was 100 to 1000 to 1, depending on the preparation that was examined.
  • the adenovirus type 5 helper virus used in the present invention was obtained by piaquer cleaning. After propagation in HeLa cells and purification via double CsCl gradient centrifugation, adenovirus was titrated again on HeLa cells in order to obtain the required number of plaque-forming units (PFU) per ml (PFU / ml) , Before use, a small subset of the virus stocks obtained was tested for the presence of wild-type AAV, but this was always negative. 4.
  • PFU plaque-forming units
  • the probes were labeled with digoxigenin, which is conventionally available as a kit from Boehringer Mannheim, Germany, and was used according to the manufacturer's instructions.
  • digoxigenin which is conventionally available as a kit from Boehringer Mannheim, Germany, and was used according to the manufacturer's instructions.
  • An alkaline phosphatase-conjugated anti-digoxigenin antibody was used for the detection, followed by an incubation with the substrate CDP
  • the samples were resuspended in Laemli sample buffer, which had 100 mM DTT, and then boiled for 15 min before they were analyzed in an SDS-PAGE.
  • the proteins were then blotted using either a "liquid transfer system” (Sigma, Germany) or a “semi-dry system” (Höfer, Germany).
  • the proof was provided with the rep specific antibody 303.9 and the cap-specific antibody B1 (Wistuba et al. (1997) J. Virol. 71, 1341-1353).
  • B7.2 expression was examined using a commercially available FITC-conjugated antibody which was directed against B7.2 (company Pharmingen International, USA).
  • an isotype control antibody and the staining of mock-transduced cells with a B7.2 antibody were carried out.
  • the cells were incubated with the antibody on ice for 30 min, then incubated twice in phosphate buffered saline before being analyzed.
  • the antibody staining was carried out in DIO buffer, the cells were resuspended in DIO buffer after the washing steps and subjected to FACS analysis.
  • the fluorescence was carried out with a "Beckton Dickinson FACS Vantage flow cytometer" at an extinction wavelength of 488 nm and an emission wavelength of 530 ⁇ 15 nm.
  • the proportion of positive cells was defined as the part whose fluorescence intensity was greater than 99% of the control cells.

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Abstract

La présente invention concerne des cellules hôtes destinées à empaqueter un virus adéno-associé recombiné (rAAV). Ces cellules comprennent au moins une copie d'une première construction d'assistant permettant l'expression d'au moins une protéine Rep d'AAV et au moins une copie d'une autre construction d'assistant permettant l'expression d'au moins une protéine Cap d'AAV. L'invention concerne également des constructions d'assistant permettant l'expression d'au moins une protéine Rep d'AAV et une protéine Cap d'AAV dans une cellule hôte, des constructions de vecteur présentant un ou plusieurs acides nucléiques hétérologues par rapport à l'AAV, ainsi que des procédés de préparation d'une cellule hôte destinée à empaqueter un virus adéno-associé recombiné (rAAV) et des utilisations de ladite cellule hôte pour produire un rAAV.
EP01967320A 2000-09-08 2001-09-07 Cellules hotes pour empaqueter un virus adeno-associe recombine (raav), leur procede de preparation et leur utilisation Withdrawn EP1315798A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10044384 2000-09-08
DE10044384A DE10044384A1 (de) 2000-09-08 2000-09-08 Wirtszellen zur Verpackung von rekombinantem Adeno-assoziiertem Virus (rAAV), Verfahren zu ihrer Herstellung und deren Verwendung
PCT/EP2001/010370 WO2002020748A2 (fr) 2000-09-08 2001-09-07 Cellules hotes pour empaqueter un virus adeno-associe recombine (raav), leur procede de preparation et leur utilisation

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CA2421442A1 (fr) 2003-03-06
JP2004508041A (ja) 2004-03-18
WO2002020748A2 (fr) 2002-03-14
US20040087026A1 (en) 2004-05-06
DE10044384A1 (de) 2002-04-18
DE10066104A1 (de) 2003-01-09
WO2002020748A3 (fr) 2003-03-20
WO2002020748A8 (fr) 2004-03-04

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