WO2003074686A1 - HELPER CONSTRUCTS FOR PRODUCING HYBRID rAAV PARTICLES OF VARIOUS AAV SEROTYPES - Google Patents

HELPER CONSTRUCTS FOR PRODUCING HYBRID rAAV PARTICLES OF VARIOUS AAV SEROTYPES Download PDF

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WO2003074686A1
WO2003074686A1 PCT/EP2003/002351 EP0302351W WO03074686A1 WO 2003074686 A1 WO2003074686 A1 WO 2003074686A1 EP 0302351 W EP0302351 W EP 0302351W WO 03074686 A1 WO03074686 A1 WO 03074686A1
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helper
rep
serotype
cap
aav
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PCT/EP2003/002351
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German (de)
French (fr)
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Markus HÖRER
Ralf Dubielzig
Stefan Ries
Meike Kosfeld-Bergauer
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Medigene Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
    • 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
    • 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

  • viruses have proven to be suitable for gene transfer in mammalian cells, especially in human cells.
  • retroviruses, adenoviruses or adeno-associated viruses are genetically modified in order to be able to use them as carriers (viral vectors) for the gene transfer of one or more transgenes, particularly in the context of gene therapy.
  • viral vectors An important aspect in the development of suitable viral vectors are safety aspects, since the vectors must not be harmful to humans or animals. For this reason, "replication-deficient" viruses are generally developed, ie viruses that can infect a cell and can transfer the transgenes into this cell, but are not able to multiply in these cells.
  • genes that are important for virus replication are deleted from the viral vector, for example genes that code for structural proteins.
  • the transgene or transgenes can be inserted in their place to produce larger amounts of non-reproducible viruses , as they are needed for gene therapy, so-called "helper genes" are necessary, which compensate for the lack of the genes important for virus multiplication in the cell.
  • AAV is a human virus from the parvovirus family, which is either integrated into the genome in the form of a provirus or causes a lytic infection in the presence of a suitable helper virus, eg adenovirus or He ⁇ es simplex virus. So far, AAV has not been causally linked to a known disease in humans. For this reason, AAV is of interest as a general transduction vector of mammalian cells.
  • the viral particles composed of three viral proteins, VP1, VP2 and VP3 contains a strand of viral DNA that has either one polarity (+) or the other polarity (-).
  • AAV as a viral transduction vector
  • rAAV viral transduction vector
  • One method for producing relatively large amounts of rAAV particles is the co-transfection of a eukaryotic cell with two recombinant AAV plasmids in the form of a mixture and infection with a helper virus (Chiorini, J. A et al. (1995) Human Gene Therapy, 6, 1531).
  • the first recombinant AAV construct contains one or more transgene (s) which are delimited by two ITR regions, ie are flanked (vector constructs).
  • the second recombinant AAV construct contains the AAV genes which are necessary for the production of the virus particles (rep and cap genes), but no ITR regions. Their absence is intended to prevent the rep and cap genes from being packaged in AAV particles and thus to prevent the formation of undesirable wild-type AAV.
  • suitable cells which are permissive for the recombinant AAV construct as well as for the helper virus, are first transfected with the two AAV constructs and then infected with helper viruses such as adenovirus.
  • Such permissive cells are, for example HeLa cells. Transfection and infection lead to expression of the AAV genes, the transgene (s) are replicated and the rAAV particles are packaged and assembled.
  • the rAAV particles contain the transgene (s), flanked on both sides by the ITR regions, in the form of a single-stranded DNA.
  • the helper virus replicates in these cells, which in the case of using adenoviruses as helper viruses generally leads to lysis and death of the infected cells after a few days.
  • the rAAV particles and also the helper viruses formed are partially released into the cell culture medium or remain in the lysed cells.
  • a review of the use of AAV as a general transduction vector for mammalian cells can be found, for example, in Muzyczka, N. in Current Topics in Microbiology and Immunology, 158, 97 (1992).
  • the control of the expression of the rep and cap genes is described, for example, in Snyder RO., Journal of Gene Medicine Preprint 1: (3) (1999).
  • AAV currently has more than ten different serotypes, with the sequences for AAV1, AAN2, AAV3, AAV4, AAV5 and AAV6 being published.
  • the serotypes differ both in the structure of the DNA and in the proteins encoded by the DNA.
  • AAV2 e.g. contains a linear single strand of DNA approximately 4.7 kilobases (kb) in length.
  • an AAV2 rep helper construct was used in addition to the helper construct with the AAV5 rep and AAV5 cap genes in order to test the complementation of the AAV2 and AAV5 rep and cap genes.
  • the hybrid AAV vectors described to date in the prior art ie with genes from different serotypes
  • regulatory sequences for example promoters and splice donor sites
  • the cap genes see, for example, Rabinowitz et al. 2002, J. Virol.
  • biodistribution refers to i) the distribution of the vector or ii) the expression of the Trnasgenes transported with the vector in different ⁇ ) tissue and organ types. It is therefore an object of the present invention to provide an expression system with which AAV vectors can be packaged in capsids of different serotypes and in which no replication-competent AAVs arise. This expression system is intended to be applicable to different serotypes and to eliminate the need to establish a completely new packaging system for each new AAV serotype.
  • helper construct for packaging rAAV in which
  • nucleic acids coding for the cap proteins of a serotype A are operatively linked to regulatory sequences of a second AAV serotype B, and b) the intron of the nucleic acids coding for the cap proteins of serotype A, and c) that for the cap and nucleic acids encoding Rep proteins are functionally separate.
  • the invention is based on the surprising finding that it is possible to express cap proteins of any serotype under the control of regulatory sequences of another serotype if the serotype of the cap proteins and that of the intron are identical, and at the same time the formation of replication-competent AAV particles by the functional separation of the nucleic acids coding for the Cap and Rep proteins.
  • the helper construct according to the invention it is possible to express the capsid proteins (cap proteins) of the respective serotype encoded by the cap gene in a correct ratio (see Example 2). In packaging approaches, these capsid proteins can form the formation of infectious hybrid rAAV particles can be achieved (see Example 3).
  • helper constructs according to the invention, it is possible to produce infectious AAV particles of different serotypes which contain a DNA of a serotype by means of a quick and simple exchange of the cap gene including its intron. This means that a DNA can be packaged very flexibly in AAV particles of different serotypes without great effort.
  • the functional separation of the nucleic acids coding for the Cap and Rep proteins surprisingly enables the production of rAAV on a large scale, but essentially prevents the formation of replication-competent AAV (rcAAV). If this was not prevented, the use of Rep and Cap of different serotypes in the event of a recombination could result in hybrid rcAAV, which would represent a new "wild type" AAV, the consequences of which are currently not foreseeable for patients the targeted exchange of the nucleic acids of one type coding for the cap proteins by another is facilitated in order to generate a desired different serotype.
  • the rep and cap genes can lie on the same helper construct or on different helper constructs.
  • the exchange of the cap helper construct is sufficient, but if they are on the same construct, the nucleic acid coding for the cap proteins, including the intron, can be exchanged by selecting suitable interfaces thereby changing the C-terminus of Rep proteins rt is.
  • 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. Specifically, this means for the rep and the cap -Gen - for the coding sequence in the wild-type AAV genome, the rep gene with the coding Se- sequence of the cap gene and the cap promoter (P40) overlap - that both genes no longer overlap. For example, this is achieved by duplicating both parts of the coding sequence used in common and the P40 promoter. 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, ie episomally, or a mixture of these.
  • the genes can also be located next to one another 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. This inventive concept also applies regardless of the serotypes of the cap genes and the regulatory sequences.
  • helper and vector constructs suitable for the production of rAAV were developed, in which the rep and cap genes were separated functionally. With these constructs, host cell lines were subsequently produced in the form of packaging and production cell lines.
  • the cap gene is under the control of P40 and the other homologous promoters actually assigned to the rep gene, that is to say P5 and P19 in the case of AAV2. This applies to both transient packaging and packaging using a packaging cell line based on HeLa cells, for example. / 074686
  • packaging cell lines based on HeLa cells were thus produced, for example, in which the rep and cap genes are functionally separated from AAV and the cap gene under the control of the homologous promoters, e.g. P5, P19 and P40 stands.
  • the sequence sections with the promoters P5, P19 and P40 were changed by mutagenesis in such a way that the promoter function remained intact, but no functional Rep protein could be expressed by these constructs.
  • a functional Rep protein is understood to mean that the Rep protein can perform the functions ascribed to it.
  • short Rep fragments are synthesized, but they cannot take on an important function of the Rep proteins.
  • Other mutagenesis options for inactivating the expression of the Rep protein e.g. by inactivating the start of the transcription are known to the person skilled in the art.
  • helper construct means recombinant AAV plasmids which contain the AAV rep genes and / or the AAV cap genes.
  • 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.
  • 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 nucleic acid segment or a genomic region which regulates the transcription of a gene to which it is linked.
  • Transcriptional regulatory sequences as described in the present invention include at least one transcriptionally active promoter, but can also include one or more enhancers and / or terminators of transcription. According to the invention, the term “regulatory sequence” does not include splice donor and splice acceptor sites.
  • operatively connected refers to the arrangement of two or more components. Since the components are related to each other, they are allowed to perform their functions in a coordinated manner.
  • a transcriptionally regulatory sequence or a promoter is operatively linked to the coding sequence if the transcriptionally regulatory sequence or the promoter regulates or starts the transcription of the coding sequence.
  • a promoter or enhancer operatively linked to a gene to be transcribed is generally referred to as an "eis" element to the coding sequence, but an enhancer is not necessarily in close proximity to the gene to be transcribed.
  • telomere a genetic entity that is altered from that entity found naturally.
  • adeno-associated virus it means that the virus carries nucleic acid (s) that were typically produced by a combination of cloning, restriction and / or ligation steps and that 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 used 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.
  • cap proteins means the proteins VP1, VP2 and / or VP3.
  • the AAV serotypes A and B are selected from the set of known serotypes AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
  • serotype B is AAV2.
  • the serotype A is AAV4 or AAV5
  • helper constructs whose regulatory sequences for the cap gene comprise at least the P40 promoter, particularly preferably the P5 and the P40 promoter, in particular the P5, the P19 and the P40 promoter of an AAV serotype heterologous to the cap gene.
  • the designations P5, P19 and P40 are used for all serotypes (Xiao et al. (1999) J. Virol. 73: 3994-4003; Bantel-Schaal et al. (1999) J. Virol. 73: 949-47; Chiorini et al. (1999) J. Virol. 73: 1309-19; Chiorini et al. (1997) J. Virol. 71: 6823-33); for AAV4, however, the P5 promoter was also referred to as the P7 promoter (Chiorini et al. (1997) J. Virol. 71: 6823-33).
  • cap gene which is solely under the control of the homologous P40 promoter is essentially constitutively expressed.
  • Helper constructs of this type are therefore not suitable for AAV packaging, since the strong, unregulated expression of the cap gene on the one hand leads to the formation of predominantly empty casids without an AAV genome, and on the other hand such helper constructs poorly integrated into a cellular genome because the constitutive expression of the cap gene appears to be toxic.
  • cap helper constructs in general and regardless of the serotypes of the cap genes and the regulatory sequences that these contain, in addition to the P40 promoter, a further promoter, preferably the P5 and / or P19 promoter, which together express the cap -Gens control.
  • This concept according to the invention is therefore independent of the serotypes of the cap genes and of the regulatory sequences.
  • Preferred helper constructs can additionally code for at least one functional Rep protein of an AAV serotype which is not identical to serotype A and is preferably of serotype B.
  • the helper constructs according to the invention are designed in such a way that the nucleic acid coding for the cap proteins is operatively linked to the regulatory sequences of another AAV serotype.
  • these regulatory sequences code for at least one functional Rep protein, which thus likewise originates from a serotype different from serotype A.
  • the Rep proteins in general can also be encoded by sequences that are not regulatory sequences of the cap genes in the helper construct.
  • the Rep proteins are 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 additional expression of Rep 68 only 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, using this helper construct can increase packaging efficiency due to the absence of Rep 78. Both Rep 68 and Rep 78 are expressed in the natural system by the P5 promoter.
  • Rep 78-deficient helper construct is also advantageous because, in comparison to Rep 78 in Adenovirus-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 78-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. This inventive concept also applies regardless of the serotypes of the cap genes and the regulatory sequences.
  • Rep proteins Rep 68 and Rep 40 and the cap proteins VP2 and VP3 are expressed, 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 AAV2 proteins with the exception of Rep 78 are encoded.
  • 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 AAV2 proteins with the exception of Rep 78.
  • helper construct pUC " ⁇ Re ⁇ 78 ⁇ Cap” (BS) ⁇ 37 (alias pUCdlRe ⁇ 78dlCa ⁇ (RBS) dl37) for the 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 " ⁇ Re ⁇ 78Cap” (RBS) ⁇ 37 (alias ⁇ UCdlRep78Cap (RBS) dl37) was deleted. This deletion means that functional cap proteins can no longer be expressed.
  • Another object of the invention is a system for packaging rAAV of an AAV serotype A consisting of at least one of the helper constructs according to the invention, in particular one of the helper constructs according to the invention, which does not necessarily code for Rep, and at least one helper construct coding for at least one functional rep Protein of another AAV serotype, preferably of serotype B.
  • the present invention furthermore relates to a host cell for packaging an rAAV of an AAV serotype A containing at least one copy of a helper construct according to the invention.
  • 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 insert an expression cassette for the AAV-Rep protein into their genome under the control of the natural regulatory elements to be integrated 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).
  • the helper constructs are transient, ie episomally transfected, in another they are stably integrated into the genome of the host cell.
  • AAV vectors are preferably produced by a packaging cell or a production cell.
  • a “packaging cell” refers to a cell that contains at least one helper construct, but no vector construct.
  • a “production cell” refers to a cell that contains both at least one helper construct and at least one vector construct.
  • the present invention thus also relates to a packaging or a production cell in which the helper construct (s) are integrated into the cellular genome.
  • the helper construct (s) are integrated into the cellular genome.
  • several identical or different helper constructs can be present at the same location, for example as concatems, or integrated at different locations.
  • Such a packaging or production cell may be dependent on a helper virus if AAV production requires infection with a helper virus. Such a packaging or production cell can, however, also be independent of the helper virus if the AAV production does not require infection with a helper virus.
  • Such a packaging or manufacturing cell which is independent of a helper virus, normally contains genes which are necessary for the induction of AAV production under the control of an inducible promoter. Such genes can be of viral or cellular origin. 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.
  • adenoviral helper genes are, for example, the genes E1A, E1B, E4, E2A and VA.
  • E1A 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
  • UL9 finally codes for a protein which binds the HSV origin of replication (see Weindler FW and Heilbronn R (1991) J Virol. 65. (5): 2476-83).
  • the use of the 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.
  • Other helper viruses are, for example, He ⁇ es viruses or Vaccinia viruses.
  • the invention further relates to the use of the helper constructs and cells according to the invention for the production of rAAV particles.
  • Suitable host cells are transfected stably or transiently with the helper constructs according to the invention. Subsequently or beforehand, the host cell is transfected stably or transiently with a suitable vector construct (see above). The cell is then cultivated under suitable conditions, so that rAAV particles are produced.
  • Methods for producing rAAV are known in the prior art (Hauswith WW et al. (2000) method in Enzymology 316, 743-761; Mathews LC et al. (2002) Methods in Enzymology 346, 393-413; Potter M et al . (2002) Methods in Enzymology 346, 413-430; Zolotukhin S et al. (1999) Gene Therapy 6, 973-985; WO 00/47757, MediGene AG; WO 02/20748, MediGene AG).
  • Fig. 1 Missing AAV5 VP1 expression.
  • C co-transfection of ⁇ UCp5Repdl37 and pUCp5pl9 ⁇ 40Ca ⁇ dl37
  • M Positive control of wtAAV / AdV-infected HeLa cells.
  • the infection occurred 48 h after transfection; 48 hours after infection, the cells were centrifuged off, lysed in cell lysis buffer and analyzed in the Western blot method.
  • the monoclonal antibody 303.9 was used to detect the Rep proteins.
  • the capsid proteins were detected with the monoclonal antibody B 1.
  • Figure 2 Correct AAV4 and AAV5 capsid protein expression.
  • I infection 10Rep
  • a cap-specific rabbit serum was used to detect the capsid proteins.
  • FIG. 3 shows a schematic representation of the plasmid pUCp5Repdl37.
  • FIG. 4 shows a schematic representation of the plasmid pUCdlRep78-dlCap (RBS) dl37.
  • FIG. 5 shows a schematic representation of the plasmid pUCp5pl9p40Capdl37.
  • FIG. 6 shows a schematic representation of the plasmid pBluAAV4dlCap.
  • 7 shows a schematic representation of the plasmid pBluAAV4dl-Rep78dlCap.
  • FIG. 8 shows a schematic representation of the plasmid pUCp5pl9p40AAV4Cap.
  • FIG. 10 shows a comparison of the intron sequences including the splice donor and splice acceptor sites of all known AAV serotypes AAVl -6.
  • the numbering given for each serotype begins with the beginning of the listed sequence with 1.
  • the black triangle shows the interface of the splice donor site, the white triangles that of the splice acceptor sites.
  • FIG. 11 shows a schematic representation of trans-split helper constructs and cis-split helper constructs.
  • cap helper plasmids for various serotypes here AAV4 and AAV5 were produced, which in combination with an AAV2 Rep helper plasmid, a recombinant AAV2 vector plasmid and, when infected with a helper virus, gave hybrid rAAV.
  • AAV4 and AAV5 were produced, which in combination with an AAV2 Rep helper plasmid, a recombinant AAV2 vector plasmid and, when infected with a helper virus, gave hybrid rAAV.
  • the AAV2 rep helper plasmid P5Rep was prepared by deleting a DNA fragment that contained nucleotides 2300-4170 of the AAV genome. (Ruffing et al. (1994) J. Gen. Virol. 75, 3385-3392 (Gene Bank Accession No. AF 043303). P5Rep ⁇ 37 (alias pUC ⁇ 5Re ⁇ dl37) was obtained from P5Rep by deleting the AAV bases 4461-4497 (FIG. 3).
  • the AAV2 rep helper construct ⁇ UC " ⁇ Re ⁇ 78 ⁇ Ca ⁇ ” (RBS) ⁇ 37 (alias ⁇ UC ⁇ Re ⁇ 78 ⁇ Cap (RBS) ⁇ 37 or pUCdlRep78dlCa ⁇ dl37) was obtained by deleting the nucleotides 3046 to 4149 from the helper construct pUC " ⁇ Rep78Capd ; Cloning see WO 00/47757 p.26 Z.14 to S.29 Z.5) prepared using the restriction enzyme Apal (Fig. 4).
  • the AAV2 cap helper plasmid P5P19P40Ca ⁇ (alias pUC ⁇ 5pl9p40Ca ⁇ ) was produced as follows: first, the cap gene was cut out of AAV2 with BamHI-SnaBI and cloned into the BamHI-Smal restriction sites of pUC19 in order to generate the plasmid pUCcap. The nucleotides 200-1056 of the AAV2 rep gene were then amplified by PCR and inserted into pUCcap via Xbal-BamHI in order to produce the AAV2 rep / cap helper plasmid pUC "rep / cap".
  • the AAV2 Rep 78 and Rep 68 deficient helper plasmid pUC ⁇ 5pl9Rep52 (40) p40Cap was then generated by deleting nucleotides 6522-6828 from pUC "rep / cap” using PpuMI-NruI-Nerdau.
  • the AAV2 cap helper plasmid pUCp5pl9p40Cap resulted from a further deletion of nucleotides 5620-6277 from pUCp5pl9Rep52 (40) p40Cap by means of BamHI-BstEII digestion and blunting of the fragment ends.
  • the AAV2 rep / cap helper plasmid pUC "rep / cap” (RBS) ⁇ 37 was generated as follows: First, the nucleotides 267-291 of pUC "rep / cap” (see above) were deleted by means of PCR mutagenesis to remove the plasmid to generate pUCAAVSapI. The nucleotides 2660-2696 of pUCAAVSapI were then deleted by means of PCR mutagenesis in order to produce the AAV2 rep / cap-Helfe ⁇ lasmid pUC "rep / cap” - (RBS) ⁇ 37.
  • the AAV2 cap helper plasmid pUCp5pl9p40Capdl37 (alias pUCp5 ⁇ l9p40-Cap ⁇ 37) was obtained via the exchange of nucleotides 4459-2989 (restriction enzymes BsiWI and Ndel) from pUCp5pl9p40Cap by nt 2426-3857 (Ndel-BsiUC ”) from ⁇ iBi" Rep made (Fig. 5).
  • the AAV4 rep helper plasmid pBluAAV4dlCap was prepared by deleting nt 3262-4407 (Sful-Smal) from ⁇ AAV4-2 (obtained from NIH; see Chiorini et al. (1997) J. Virol. 71: 6823-33) (Fig 6).
  • the plasmid pBluAAV4Rep68,40Cap was first prepared by deleting nt 2445-2770 from pAAV4-2 via PCR-based mutagenesis.
  • the nucleotides 3906-4498 (Xbal-Mlul) from pBlu-AAV4dlCap were then replaced by nt 677-2937 (Xbal-Sfol) from pBluAAV4Rep68.40Cap (FIG. 7).
  • a NotI interface was first inserted at position nt 3156 of pUCp5pl9p40Capdl37 by PCR-based mutagenesis in order to generate the intermediate product pUCp5pl9p40Capdl37Not2.
  • the nucleotides 348-3156 (Ndel-Notl) of pUC ⁇ 5pl9p40Ca ⁇ dl37Not2 were then replaced by the PCR-amplified nt 2365-5164 from ⁇ AAV4-2 via Notl-Ndel (FIG. 8).
  • the AAV5 cap helper plasmid pUCp5pl9p40AAV5Cap was obtained by replacing the nucleotides 348-3156 (Ndel-Notl) of pUCp5pl9 ⁇ 40Ca ⁇ dl37Not2 with the PCR-amplified nt 2305-5095 from pAAV5-2 (obtained from NIH; see Chiorini J. et al. (1999) Virol.
  • Hybrid rAAV were produced in HeLa cells after co-transfection of pUCp5Repdl37 and pUC ⁇ 5pl9p40AAV5Ca ⁇ dl37 clones 1 and 2 as well as adenovirus (AdV) infection (MOI 10) according to standard methods (see e.g. WO 00/47757).
  • AdV adenovirus infection
  • the coding sequences of the AAV5 cap gene are under the control of the regulatory sequences of AAV2, these AAV2 sequences comprising the AAV2 cap splice donor site.
  • splice donor and splice acceptor sites are derived from different serotypes, AAV2 and AAV5.
  • Hybrid rAAV were produced in HeLa cells after cotransfection of pUCp5pl9p40AAV4Cap (4Cap) or pUCp5pl9p40AAV5Cap (5Cap) and pBluAAV4dlRep78dlCap (4Rep) or pUCdlRep78dlCapdl37 (MOI10D) and infection (MOI 10Dep).

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Abstract

The invention relates to helper constructs for rAAV packaging, in which the nucleic acids coding for the Cap proteins of a serotype A are operatively bound to regulatory sequences of a second AAV serotype B, the intervening sequence of the nucleic acids coding for the Cap proteins is of serotype A, and the nucleic acids coding for the Cap and Rep proteins are functionally separated.

Description

Helferkonstrukte für die Herstellung hybrider rAAV-Partikel unterschiedlicher AAV-Serotypen Helper constructs for the production of hybrid rAAV particles of different AAV serotypes
Genetisch modifizierte Viren haben sich für den Gentransfer in Säugerzellen, insbesondere in humane Zellen als geeignet herausgestellt. Zurzeit werden unter anderem Retroviren, Adenoviren oder adeno-assoziierte Viren (AAV) genetisch modifiziert, um sie als Träger (virale Vektoren) für den Gentransfer eines oder mehrerer Transgene insbesondere im Rahmen einer Gentherapie verwenden zu können. Ein wesentlicher Gesichtspunkt bei der Entwicklung geeigneter viraler Vektoren sind Sicherheitsaspekte, da die Vektoren für den Menschen oder das Tier nicht schädlich sein dürfen. Aus diesem Grund werden im Allgemeinen „replikationsdefiziente" Viren entwickelt, d.h. Viren, die zwar eine Zelle infizieren und die Transgene in diese Zelle transferieren können, die aber nicht in der Lage sind, sich in diesen Zellen zu vermehren. Dies wird zum Beispiel dadurch erreicht, dass Gene, die für die Virusvermehrung wichtig sind, aus dem viralen Vektor deletiert werden. Dies sind beispielsweise Gene, die für Strukturproteine kodieren. An deren Stelle können das Transgen oder die Transgene eingebaut werden. Zur Produktion von größeren Mengen an nicht-vermehrbaren Viren, wie sie für die Gentherapie benötigt werden, sind sog. „Helfergene" notwendig, die das Fehlen der für die Virusvermehrung wichtigen Gene in der Zelle kompensieren.Genetically modified viruses have proven to be suitable for gene transfer in mammalian cells, especially in human cells. Currently, among other things, retroviruses, adenoviruses or adeno-associated viruses (AAV) are genetically modified in order to be able to use them as carriers (viral vectors) for the gene transfer of one or more transgenes, particularly in the context of gene therapy. An important aspect in the development of suitable viral vectors are safety aspects, since the vectors must not be harmful to humans or animals. For this reason, "replication-deficient" viruses are generally developed, ie viruses that can infect a cell and can transfer the transgenes into this cell, but are not able to multiply in these cells. This is achieved, for example, by this that genes that are important for virus replication are deleted from the viral vector, for example genes that code for structural proteins. The transgene or transgenes can be inserted in their place to produce larger amounts of non-reproducible viruses , as they are needed for gene therapy, so-called "helper genes" are necessary, which compensate for the lack of the genes important for virus multiplication in the cell.
AAV ist ein humanes Virus aus der Familie der Parvoviren, welches entweder in Form eines Provirus in das Genom integriert vorliegt oder in Anwesenheit eines geeigneten Helfervirus, z.B. Adenovirus oder Heφes Simplex Virus, eine lytische Infektion verursacht. AAV konnte bisher in keinen kausalen Zusammenhang mit einer bekannten Erkrankung beim Menschen gebracht werden. Aus diesem Grund ist AAV als allgemeiner Transduktionsvektor von Säugerzellen von Interesse. Die viralen Partikel, die aus drei viralen Proteinen, VP1, VP2 und VP3 zusammenge- setzt sind, enthalten einen Strang viraler DNA, welcher entweder die eine Polarität (+) oder die andere Polarität (-) besitzt.AAV is a human virus from the parvovirus family, which is either integrated into the genome in the form of a provirus or causes a lytic infection in the presence of a suitable helper virus, eg adenovirus or Heφes simplex virus. So far, AAV has not been causally linked to a known disease in humans. For this reason, AAV is of interest as a general transduction vector of mammalian cells. The viral particles composed of three viral proteins, VP1, VP2 and VP3 contains a strand of viral DNA that has either one polarity (+) or the other polarity (-).
Die Verwendung von AAV als viralen Transduktionsvektor (rekombinanter AAV, rAAV) erfordert im allgemeinen relativ große Mengen an rekombinanten AAV- Partikeln. Eine Methode zur Herstellung relativ großer Mengen an rAAV- Partikeln ist die Co-Transfektion einer eukaryotischen Zelle mit zwei rekombinanten AAV-Plasmiden in Form einer Mischung und Infektion mit einem Helfervirus (Chiorini, J. A et al. (1995) Human Gene Therapy, 6, 1531). Das erste re- kombinante AAV-Konstrukt enthält ein oder mehrere Transgen(e), welche(s) von zwei ITR-Regionen begrenzt, d.h. flankiert wird (werden) (Vektorkonstrukte). Das zweite rekombinante AAV-Konstrukt, das Helfer-Konstrukt, enthält die AAV-Gene, welche für die Herstellung der Viruspartikel notwendig sind (rep- und cap-Gene), aber keine ITR-Regionen. Deren Abwesenheit soll die Verpa- ckung der rep- und cap-Gene in AAV-Partikel und damit die Bildung von unerwünschtem Wildtyp AAV verhindern. Zur Herstellung von AAV werden geeignete Zellen, welche sowohl für das rekombinante AAV-Konstrukt, als auch für das Helfervirus permissiv, d.h. zugänglich sind, zunächst mit den beiden AAV- Konstrukten transfiziert und dann mit Helferviren wie z.B. Adenovirus infiziert, Solche permissiven Zellen sind z.B. HeLa-Zellen. Transfektion und Infektion führen zur Expression der AAV-Gene, die (das) Transgen(e) werden repliziert und die rAAV-Partikel werden verpackt und zusammengesetzt. Die rAAV-Partikel enthalten das (die) Transgen(e), auf beiden Seiten flankiert durch die ITR- Regionen, in Form einer einzelsträngigen DNA. Zur gleichen Zeit repliziert das Helfervirus in diesen Zellen, was im Fall der Verwendung von Adenoviren als Helferviren im allgemeinen zu Lyse und Tod der infizierten Zellen nach wenigen Tagen führt. Die rAAV-Partikel und auch die gebildeten Helferviren werden hierbei teilweise in das Zellkulturmedium freigesetzt oder verbleiben in den lysierten Zellen. Ein Review über die Verwendung von AAV als allgemeinen Transdukti- onsvektor für Säugerzellen findet sich z.B. bei Muzyczka, N. in Current Topics in Microbiology and Immunology, 158, 97 (1992). Die Steuerung der Expression der rep- und cap-Gene wird beispielsweise in Snyder RO., Journal of Gene Medicine Preprint 1:(3) (1999) beschrieben.The use of AAV as a viral transduction vector (recombinant AAV, rAAV) generally requires relatively large amounts of recombinant AAV particles. One method for producing relatively large amounts of rAAV particles is the co-transfection of a eukaryotic cell with two recombinant AAV plasmids in the form of a mixture and infection with a helper virus (Chiorini, J. A et al. (1995) Human Gene Therapy, 6, 1531). The first recombinant AAV construct contains one or more transgene (s) which are delimited by two ITR regions, ie are flanked (vector constructs). The second recombinant AAV construct, the helper construct, contains the AAV genes which are necessary for the production of the virus particles (rep and cap genes), but no ITR regions. Their absence is intended to prevent the rep and cap genes from being packaged in AAV particles and thus to prevent the formation of undesirable wild-type AAV. For the production of AAV, suitable cells, which are permissive for the recombinant AAV construct as well as for the helper virus, are first transfected with the two AAV constructs and then infected with helper viruses such as adenovirus. Such permissive cells are, for example HeLa cells. Transfection and infection lead to expression of the AAV genes, the transgene (s) are replicated and the rAAV particles are packaged and assembled. The rAAV particles contain the transgene (s), flanked on both sides by the ITR regions, in the form of a single-stranded DNA. At the same time, the helper virus replicates in these cells, which in the case of using adenoviruses as helper viruses generally leads to lysis and death of the infected cells after a few days. The rAAV particles and also the helper viruses formed are partially released into the cell culture medium or remain in the lysed cells. A review of the use of AAV as a general transduction vector for mammalian cells can be found, for example, in Muzyczka, N. in Current Topics in Microbiology and Immunology, 158, 97 (1992). The control of the expression of the rep and cap genes is described, for example, in Snyder RO., Journal of Gene Medicine Preprint 1: (3) (1999).
Von AAV sind zur Zeit mehr als zehn verschiedene Serotypen bekannt, wobei für AAV1, AAN2, AAV3, AAV4, AAV5 und AAV6 die Sequenzen publiziert sind. Die Serotypen unterscheiden sich sowohl im Aufbau der DNA als auch in den durch die DNA kodierten Proteine. AAV2 z.B. enthält eine lineare Einzelstrang DNA von ca. 4,7 Kilobasen (kb) Länge.AAV currently has more than ten different serotypes, with the sequences for AAV1, AAN2, AAV3, AAV4, AAV5 and AAV6 being published. The serotypes differ both in the structure of the DNA and in the proteins encoded by the DNA. AAV2 e.g. contains a linear single strand of DNA approximately 4.7 kilobases (kb) in length.
Im Stand der Technik ist bekannt, dass bestimmte Gewebetypen von einzelnen AAV Serotypen unterschiedlich transduziert werden (z.B. Davidson et al. (2000) Proc. Natl. Acad. Sei. 97:3428-32; Duan et al. (2001) J. Virol. 75:7662-71). Somit ist es vorteilhaft, im Rahmen einer Gentherapie für jeden zu transfizierenden Gewebetyp einen unterschiedlichen AAV-Serotyp zu verwenden. Dies hat jedoch den Nachteil, dass für jeden Serotyp eine eigenes Transduktionssystem etabliert werden muss.It is known in the prior art that certain tissue types are transduced differently from individual AAV serotypes (for example Davidson et al. (2000) Proc. Natl. Acad. Sci. 97: 3428-32; Duan et al. (2001) J. Virol 75: 7662-71). It is therefore advantageous to use a different AAV serotype for each tissue type to be transfected as part of gene therapy. However, this has the disadvantage that a separate transduction system must be established for each serotype.
Im Stand der Technik wurde bereits gezeigt, dass es möglich ist, rekombinante AAV2- Vektoren in Kapside anderer Serotypen zu verpacken, und zwar unter Verwendung der AAV2 Rep-Proteine (Chiorini et al. 1999 J. Virol. 73:1309-19; Xiao et al. 1999 J. Virol. 73:3994-4003; Rabinowitz et al. 2002 J. Virol. 76:791- 801). Dabei wurden in der Regel hybride Helferkonstrukte verwendet, die das rep- Gen von AAV2 und das cap-Gen des anderen Serotyps enthielten. Lediglich bei Chiorini et al. (1999) wurde in Ermangelung eines hybriden Helferkonstrukts zusätzlich zu dem Helferkonstrukt mit dem AAV5 rep- und dem AAV5 cap-Gen ein AAV2 rep-Helferkonstrukt eingesetzt, um die Komplementation der AAV2 und AAV5 rep- und cap-Gene zu testen. Die bislang im Stand der Technik beschriebenen hybriden (d.h. mit Genen aus verschiedenen Serotypen) AAV- Vektoren haben mit einer Ausnahme gemein, dass zur Expression der cap-Gene regulatorische Sequenzen (z.B. Promotoren und Spleissdonorstellen) verwendet wurden, die vom gleichen Serotyp stammen (s. z.B. Rabinowitz et al. 2002, J. Virol. 76:791-801, wo die Hypothese aufgestellt wurde, dass eine Rep-spezifische Kapsid-Interaktionsdomäne für eine effiziente Serotyp-spezifische Enkapsidierung benötigt würde). Dies hat jedoch den Nachteil, dass für jeden Serotyp ein eigenes Cap-Expressionssystem etabliert werden muss, das aus regulatorischen Sequenzen und für Cap-Proteine kodierenden Se- quenzen insgesamt eine Serotyps besteht. Bei derartigen Expressionssystemen kann nicht vorausgesagt werden, ob es zusammen mit den Rep-Proteinen des anderen Serotyps zu infektiösen AAV-Partikeln führt.It has already been shown in the prior art that it is possible to package recombinant AAV2 vectors in capsids of other serotypes, using the AAV2 Rep proteins (Chiorini et al. 1999 J. Virol. 73: 1309-19; Xiao et al. 1999 J. Virol. 73: 3994-4003; Rabinowitz et al. 2002 J. Virol. 76: 791-801). Hybrid helper constructs containing the rep gene of AAV2 and the cap gene of the other serotype were generally used. Only in Chiorini et al. (1999), in the absence of a hybrid helper construct, an AAV2 rep helper construct was used in addition to the helper construct with the AAV5 rep and AAV5 cap genes in order to test the complementation of the AAV2 and AAV5 rep and cap genes. The hybrid AAV vectors described to date in the prior art (ie with genes from different serotypes) have one thing in common, with the exception that regulatory sequences (for example promoters and splice donor sites) which originate from the same serotype were used to express the cap genes (see, for example, Rabinowitz et al. 2002, J. Virol. 76: 791-801, where it was hypothesized that a Rep-specific capsid interaction domain would be required for efficient serotype-specific encapsidation). However, this has the disadvantage that a separate cap expression system must be established for each serotype, which consists of regulatory sequences and a serotype encoding sequences for cap proteins. With such expression systems, it cannot be predicted whether, together with the Rep proteins of the other serotype, it leads to infectious AAV particles.
In der Publikation Xiao et al. ((1999) J. Virol. 73:3994-4003) wird ein AAV Rep/Cap-Helferplasmid beschrieben, bei dem das cap-Gen von AAVl unter der Kontrolle des p40-Promotors des AAV-Serotyps steht, dessen rep-Gen verwendet wurde (AAV2). Dieses System ist zwar prinzipiell zur Generierung von hybriden AAV-Partikeln geeignet, birgt aber die Gefahr, dass durch ein einziges Rekombinationsereignis mit einem Vektorkonstrukt hybride replikationskompetente AAV- Partikel entstehen können. Die Generierung derartiger Partikel ist zu vermeiden, da mögliche negative Auswirkungen solcher neuer Virustypen für Mensch und Tier zunächst nicht ausgeschlossen werden können und die Gefahr eines „Vector- Shedding" (Ausscheiden von Vektor über Speichel, Urin etc.) bzw. einer unerwünschten Bioverteilung erhöht ist.In the publication Xiao et al. ((1999) J. Virol. 73: 3994-4003) describes an AAV Rep / Cap helper plasmid in which the cap gene of AAV1 is under the control of the p40 promoter of the AAV serotype, whose rep gene is used was (AAV2). Although this system is principally suitable for generating hybrid AAV particles, it harbors the risk that hybrid replication-competent AAV particles can arise from a single recombination event with a vector construct. The generation of such particles should be avoided since possible negative effects of such new virus types for humans and animals cannot be ruled out initially and the risk of "vector shedding" (excretion of vector via saliva, urine, etc.) or undesired biodistribution increases is.
Der Begriff "Bioverteilung" bezieht sich auf i) die Verteilung des Vektors auf oder ii) die Expression der mit dem Vektor tranportierten Trnasgene in verschiedene^) Gewebe- und Organtypen. Aufgabe der vorliegenden Erfindung ist es daher, ein Expressionssystem bereitzustellen, mit dem AAV- Vektoren in Kapside unterschiedlicher Serotypen verpackt werden können und bei dem möglichst keine replikationskompetenten AAV entstehen. Dieses Expressionssystem soll für verschiedene Serotypen anwendbar sein, und die Notwendigkeit beseitigen, bei jedem neuen AAV-Serotyp ein komplett neues Verpackungssystem zu etablieren.The term "biodistribution" refers to i) the distribution of the vector or ii) the expression of the Trnasgenes transported with the vector in different ^) tissue and organ types. It is therefore an object of the present invention to provide an expression system with which AAV vectors can be packaged in capsids of different serotypes and in which no replication-competent AAVs arise. This expression system is intended to be applicable to different serotypes and to eliminate the need to establish a completely new packaging system for each new AAV serotype.
Erfindungsgemäß wird die Aufgabe durch ein Helferkonstrukt zur Verpackung von rAAV gelöst, bei demAccording to the invention, the object is achieved by a helper construct for packaging rAAV, in which
a) die für die Cap-Proteine eines Serotyps A kodierenden Nukleinsäuren operativ mit regulatorischen Sequenzen eines zweiten AAV Serotyps B verbunden sind, und b) das Intron der für die Cap-Proteine kodierenden Nukleinsäuren vom Serotyp A ist, und c) die für die Cap- und Rep-Proteine kodierenden Nukleinsäuren funktio- nell getrennt vorliegen.a) the nucleic acids coding for the cap proteins of a serotype A are operatively linked to regulatory sequences of a second AAV serotype B, and b) the intron of the nucleic acids coding for the cap proteins of serotype A, and c) that for the cap and nucleic acids encoding Rep proteins are functionally separate.
Der Erfindung Hegt der überraschende Befund zu Grunde, dass es möglich ist, Cap-Proteine eines beliebigen Serotyps unter der Kontrolle regulatorischer Sequenzen eines anderen Serotyps zu exprimieren, wenn der Serotyp der Cap- Proteine und derjenige des Introns identisch ist, und dabei gleichzeitig die Entstehung von replikationskompetenten AAV-Partikeln durch die funktionelle Trennung der für die Cap- und Rep-Proteine kodierenden Nukleinsäuren zu verhin- dern. Mit dem erfindungsgemäßen Helferkonstrukt ist es möglich, die durch das cap-Gen kodierten Kapsidproteine (Cap-Proteine) des jeweiligen Serotyps in einem korrekten Verhältnis zu exprimieren (s. Beispiel 2). In Verpackungsansätzen kann mit diesen Kapsidproteinen die Bildung infektiöser hybrider rAAV-Partikel erzielt werden (s. Beispiel 3). Ferner ist es mit Hilfe der erfindungsgemäßen Hel- ferkonstrukte möglich, durch einen schnellen und einfachen Austausch des cap- Genes einschließlich seines Introns infektiöse AAV-Partikel verschiedener Serotypen herzustellen, die eine DNA eines Serotyps enthalten. Somit kann ohne gro- ßen Aufwand sehr flexibel eine DNA in AAV-Partikel verschiedener Serotypen verpackt werden.The invention is based on the surprising finding that it is possible to express cap proteins of any serotype under the control of regulatory sequences of another serotype if the serotype of the cap proteins and that of the intron are identical, and at the same time the formation of replication-competent AAV particles by the functional separation of the nucleic acids coding for the Cap and Rep proteins. With the helper construct according to the invention, it is possible to express the capsid proteins (cap proteins) of the respective serotype encoded by the cap gene in a correct ratio (see Example 2). In packaging approaches, these capsid proteins can form the formation of infectious hybrid rAAV particles can be achieved (see Example 3). Furthermore, with the aid of the helper constructs according to the invention, it is possible to produce infectious AAV particles of different serotypes which contain a DNA of a serotype by means of a quick and simple exchange of the cap gene including its intron. This means that a DNA can be packaged very flexibly in AAV particles of different serotypes without great effort.
Durch die fünktionelle Trennung der für die Cap- und Rep-Proteine kodierenden Nukleinsäuren gelingt es überraschenderweise, die Produktion von rAAV in gro- ßem Maßstab zu erlauben, dabei aber die Entstehung von replikationskompeten- tem AAV (rcAAV) im Wesentlichen zu verhindern. Wäre diese nicht verhindert, so könnten durch die Verwendung von Rep und Cap verschiedener Serotypen im Falle einer Rekombination hybride rcAAV entstehen, die einen neuen „Wildtyp" - AAV darstellen würden, dessen Folgen für Patienten derzeit nicht absehbar sind. Zugleich wird durch die funktioneile Trennung der gezielte Austausch der für die Cap-Proteine kodierenden Nukleinsäuren eines Typs durch die eines anderen erleichtert, um einen gewünschten anderen Serotyp zu generieren. Erfmdungsgemäß können dabei die rep- und cap-Gene auf demselben Helferkonstrukt oder auf unterschiedlichen Helferkonstrukten liegen. Liegen die rep- und cap-Gene auf unter- schiedlichen Helferkonstrukten, so genügt der Austausch des cap-Helfer- konstrukts. Liegen sie hingegen auf demselben Konstrukt, so kann durch Wahl geeigneter Schnittstellen die für die Cap-Proteine kodierende Nukleinsäure einschließlich des Introns ausgetauscht werden, ohne dass dabei der C-Terminus von Rep-Proteinen verändert wird.The functional separation of the nucleic acids coding for the Cap and Rep proteins surprisingly enables the production of rAAV on a large scale, but essentially prevents the formation of replication-competent AAV (rcAAV). If this was not prevented, the use of Rep and Cap of different serotypes in the event of a recombination could result in hybrid rcAAV, which would represent a new "wild type" AAV, the consequences of which are currently not foreseeable for patients the targeted exchange of the nucleic acids of one type coding for the cap proteins by another is facilitated in order to generate a desired different serotype. According to the invention, the rep and cap genes can lie on the same helper construct or on different helper constructs. and cap genes on different helper constructs, the exchange of the cap helper construct is sufficient, but if they are on the same construct, the nucleic acid coding for the cap proteins, including the intron, can be exchanged by selecting suitable interfaces thereby changing the C-terminus of Rep proteins rt is.
Unter den Ausdrücken „fünktionell unabhängige Einheiten" oder „funktionell getrennt" wird verstanden, dass zwei oder mehrere Gene nicht überlappen, wobei der Begriff „Gen" neben der kodierenden Sequenz auch den entsprechenden Promotor umfasst. Konkret heißt dies für das rep- und das cap-Gen - für die im Wild- typ AAV-Genom die kodierende Sequenz das rep-Gens mit der kodierenden Se- quenz des cap-Gens und dem cap-Promotor (P40) überlappt -, dass beide Gene nicht mehr überlappen. Beispielsweise gelingt dies dadurch, dass beide Teile der gemeinsam verwendeten kodierenden Sequenz und der P40 Promotor dupliziert werden. Dies kann verschiedene Anordnungen der Gene in einem Genom bedeu- ten. Zum einen können die Gene an verschiedenen Orten im Genom lokalisiert sein, sei es an verschiedenen Stellen in das Genom integriert oder auf unterschiedlichen Plasmiden, also episomal, lokalisiert oder eine Mischung aus diesen. Zum anderen können die Gene auch nebeneinander auf demselben DNA-Molekül, beispielsweise einem Chromosom oder einem Plasmid, lokalisiert sein, wobei jedoch jedes Gen von seinem eigenen Promotor aus kontrolliert wird. Eine derartige Anordnung ist zum Beispiel dann wahrscheinlich, wenn zwei Gene auf unterschiedlichen DNA-Molekülen gemeinsam transfiziert werden. Diese Moleküle können während der Transfektion Konkatemere bilden, die dann an einer Stelle in das Genom integrieren, jedoch nach wie vor funktioneil unabhängige Einheiten bil- den. Dieses erfinderische Konzept gilt auch unabhängig von den Serotypen der cap-Gene sowie den regulatorischen Sequenzen.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. Specifically, this means for the rep and the cap -Gen - for the coding sequence in the wild-type AAV genome, the rep gene with the coding Se- sequence of the cap gene and the cap promoter (P40) overlap - that both genes no longer overlap. For example, this is achieved by duplicating both parts of the coding sequence used in common and the P40 promoter. This can mean different arrangements of the genes in a genome. On the one hand, 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, ie episomally, or a mixture of these. On the other hand, the genes can also be located next to one another 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. This inventive concept also applies regardless of the serotypes of the cap genes and the regulatory sequences.
Hierzu wurden neue, für die Herstellung von rAAV geeignete Helfer- und Vek- torkonstrukte entwickelt, in denen die rep- und cap-Gene funktioneil getrennt wurden. Mit diesen Konstrukten wurden anschließend Wirtszelllinien in Form von Verpackungs- und Produktionszelllinien hergestellt.For this purpose, new helper and vector constructs suitable for the production of rAAV were developed, in which the rep and cap genes were separated functionally. With these constructs, host cell lines were subsequently produced in the form of packaging and production cell lines.
In einer bevorzugten Ausführungsform befindet sich das cap-Gen unter der Kontrolle von P40 und der anderen, eigentlich dem rep-Gen zugeordneten, homologen Promotoren, im Falle von AAV2 also P5 und P19. Dies gilt sowohl bei transien- ten Verpackungen als auch bei der Verpackung mit Hilfe einer beispielsweise auf HeLa-Zellen basierten Verpackungszelllinie. /074686In a preferred embodiment, the cap gene is under the control of P40 and the other homologous promoters actually assigned to the rep gene, that is to say P5 and P19 in the case of AAV2. This applies to both transient packaging and packaging using a packaging cell line based on HeLa cells, for example. / 074686
Mit den beschriebenen Konstrukten wurden somit beispielsweise auf HeLa-Zellen basierende Verpackungszelllinien hergestellt, in denen die rep- und cap-Gene von AAV funktionell getrennt sind sowie das cap-Gen unter der Kontrolle der homologen Promotoren, z.B. P5, P19 und P40 steht. Dabei wurden beispielsweise für die AAV2 Helferkonstrukte die Sequenzabschnitte mit den Promotoren P5, P19 und P40 durch Mutagenese derart verändert, dass zwar die Promotorfunktion intakt blieb, durch diese Konstrukte aber kein funktionelles Rep-Protein exprimiert werden konnte. Unter einem fünktionellen Rep-Protein wird in diesem Zusammenhang verstanden, dass das Rep-Protein seine ihm zugeschriebenen Funktionen ausüben kann. Im vorliegenden Fall werden zwar kurze Rep-Fragmente synthetisiert, die aber keine wichtige Funktion der Rep-Proteine übernehmen können. Andere Mutagenese-Möglichkeiten zur Inaktivierung der Expression des Rep- Proteins, z.B. durch die Inaktivierung des Starts der Transkription, sind dem Fachmann bekannt.With the constructs described, packaging cell lines based on HeLa cells were thus produced, for example, in which the rep and cap genes are functionally separated from AAV and the cap gene under the control of the homologous promoters, e.g. P5, P19 and P40 stands. For example, for the AAV2 helper constructs, the sequence sections with the promoters P5, P19 and P40 were changed by mutagenesis in such a way that the promoter function remained intact, but no functional Rep protein could be expressed by these constructs. In this context, a functional Rep protein is understood to mean that the Rep protein can perform the functions ascribed to it. In the present case, short Rep fragments are synthesized, but they cannot take on an important function of the Rep proteins. Other mutagenesis options for inactivating the expression of the Rep protein, e.g. by inactivating the start of the transcription are known to the person skilled in the art.
Für die Helfervirus-induzierbare Transaktivierung des Promotors P40 mussten die beiden großen Rep-Proteine Rep 68 und Rep 78 von einer zweiten Quelle (in trans oder in eis) zur Verfügung gestellt werden. Mit Hilfe solcher Cap-Expressions- konstrukte konnte nun die stabile Integration in das Genom der Wirtszellen er- reicht werden. Diese Konstrukte besitzen somit den Vorteil, dass in Abwesenheit eines Helfervirus keine toxischen Mengen an Cap-Proteinen bzw. ein Überschuss an leeren Kapsiden gebildet werden und dennoch eine sehr starke, durch Helferviren induzierbare Cap-Proteinexpression gewährleistet ist.For the helper virus-inducible transactivation of the promoter P40, the two large Rep proteins Rep 68 and Rep 78 had to be provided by a second source (in trans or in eis). With the help of such cap expression constructs, stable integration into the genome of the host cells could now be achieved. These constructs thus have the advantage that, in the absence of a helper virus, no toxic amounts of cap proteins or an excess of empty capsids are formed, and nevertheless a very strong cap protein expression which can be induced by helper viruses is ensured.
Unter dem Begriff „Helferkonstrukt" werden im Rahmen der vorliegenden Erfindung rekombinante AAV-Plasmide verstanden, die die AAV rep-Gene und/oder die AAV cap-Gene enthalten. Die Begriffe „Protein" und „Polypeptid" werden im Rahmen der vorliegenden Erfindung synonym gebraucht und beziehen sich auf ein Polymer von Aminosäuren beliebiger Länge. Diese Begriffe schließen ebenso Proteine mit ein, die post- translationale Modifikationsschritte durchlaufen haben, wie beispielsweise Glyko- sylierung, Acetylierung oder Phosphorylierung.In the context of the present invention, the term “helper construct” means recombinant AAV plasmids which contain the AAV rep genes and / or the AAV cap genes. The terms “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.
Unter den Begriffen „Nukleinsäure", „DNA" und „Polynukleotide" im Sinne der vorliegenden Erfindung sind polymere Formen von Nukleotiden jeder Länge zu verstehen, wobei sich der Begriff nur auf die Primärstruktur des Moleküls bezieht. Daher sind einzel- und doppelsträngige DNA-Moleküle ebenso von diesem Begriff umfasst wie modifizierte Polynukleotide wie beispielsweise methylierte oder geschützte (= capped) Polynukleotide.The terms “nucleic acid”, “DNA” and “polynucleotides” in the context of the present invention are to be understood as meaning polymeric forms of nucleotides of any length, the term only referring to the primary structure of the molecule. Therefore, single and double-stranded DNA molecules this term also includes modified polynucleotides such as methylated or protected (= capped) polynucleotides.
Die Begriffe „Gene" bzw. „Gensequenzen" beziehen sich auf ein Polynukleotid, das mindestens einen offenen Leserahmen aufweist und das die Fähigkeit besitzt, durch Transkription und Translation ein bestimmtes Protein zu bilden.The terms “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.
Unter dem Begriff „regulatorische Sequenz" wird ein Nukleinsäureabschnitt oder eine genomische Region verstanden, welche(r) die Transkription eines Gens, mit dem es verbunden ist, reguliert. Transkriptioneil regulatorische Sequenzen, so wie sie in der vorliegenden Erfindung beschrieben sind, schließen wenigstens einen transkriptionell aktiven Promotor ein, können aber auch einen oder mehrere En- hancer und/oder Terminatoren der Transkription umfassen. Erfindungsgemäß umfasst der Begriff „regulatorische Sequenz" nicht Spleißdonor- und Spleißakzep- torstellen.The term "regulatory sequence" is understood to mean a nucleic acid segment or a genomic region which regulates the transcription of a gene to which it is linked. Transcriptional regulatory sequences as described in the present invention include at least one transcriptionally active promoter, but can also include one or more enhancers and / or terminators of transcription. According to the invention, the term “regulatory sequence” does not include splice donor and splice acceptor sites.
Der Begriff „operativ verbunden" bezieht sich auf die Anordnung von zwei oder mehr Komponenten. Da die Komponenten in einer Beziehung zueinander stehen, wird ihnen erlaubt, ihre Funktion in einer koordinierten Weise auszuüben. Beispielsweise ist eine transkriptioneil regulatorische Sequenz oder ein Promotor operativ mit der kodierenden Sequenz verbunden, wenn die transkriptionell regulatorische Sequenz bzw. der Promotor die Transkription der kodierenden Sequenz reguliert bzw. startet. Ein operativ mit einem zu transkribierenden Gen verbundener Promotor oder Enhancer wird generell als „eis" -Element zu der kodierenden Sequenz bezeichnet, aber ein Enhancer befindet sich nicht notwendigerweise in direkter räumlicher Nähe zu dem zu transkribierenden Gen.The term "operatively connected" refers to the arrangement of two or more components. Since the components are related to each other, they are allowed to perform their functions in a coordinated manner. For example, a transcriptionally regulatory sequence or a promoter is operatively linked to the coding sequence if the transcriptionally regulatory sequence or the promoter regulates or starts the transcription of the coding sequence. A promoter or enhancer operatively linked to a gene to be transcribed is generally referred to as an "eis" element to the coding sequence, but an enhancer is not necessarily in close proximity to the gene to be transcribed.
Der Ausdruck „rekombinant", so wie er im Rahmen dieser Erfindung gebraucht ist, bezieht sich auf eine genetische Einheit, die gegenüber jener Einheit, die natürlicherweise gefunden wird, verändert ist. Wenn der Begriff auf ein Adeno- assoziiertes Virus angewendet wird, bedeutet dies, dass das Virus Nukleinsäure/n trägt, die typischerweise durch eine Kombination von Klonierungs-, Restriktions- und/oder Ligationsschritten hergestellt wurde/n und die natürlicherweise nicht in dem Adeno-assoziierten Virus vorkommt/vorkommen.The term "recombinant" as used in the context of this invention refers to a genetic entity that is altered from that entity found naturally. When the term is applied to an adeno-associated virus, it means that the virus carries nucleic acid (s) that were typically produced by a combination of cloning, restriction and / or ligation steps and that do not naturally occur in the adeno-associated virus.
Die Begriffe „natürlicher Promotor" bzw. „homologer Promotor", so wie sie im Rahmen der Erfindung gebraucht werden, bedeuten, dass die genetische Einheit des Promotors bzw. der regulatorischen Sequenz aus dem gleichen Organismus stammt wie der Rest der Einheit, mit dem sie verglichen wird. Umgekehrt bedeutet ein „heterologer" oder „nicht natürlicher Promotor", dass der Promotor von seiner natürlichen kodierenden Sequenz getrennt wurde und operativ mit einer anderen kodierenden Sequenz verbunden wurde.The terms “natural 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 used 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.
Erfindungsgemäß werden unter dem Begriff „Cap-Proteine" die Proteine VP1, VP2 und/oder VP3 verstanden. Gemäß einer bevorzugten Ausführungsform sind die AAV-Serotypen A und B ausgewählt aus der Menge der bekannten Serotypen AAVl, AAV2, AAV3, AAV4, AAV5 und AAV6.According to the invention, the term “cap proteins” means the proteins VP1, VP2 and / or VP3. According to a preferred embodiment, the AAV serotypes A and B are selected from the set of known serotypes AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
Gemäß einer besonders bevorzugten Ausführungsform ist der Serotyp B AAV2.According to a particularly preferred embodiment, serotype B is AAV2.
Gemäß einer besonders bevorzugten Ausführungsform ist der Serotyp A AAV4 oder AAV5According to a particularly preferred embodiment, the serotype A is AAV4 or AAV5
Ferner sind solche Helferkonstrukte bevorzugt, deren regulatorische Sequenzen für das cap-Gen mindestens den P40 Promotor, besonders bevorzugt den P5 und den P40 Promotor, insbesondere den P5, den P19 und den P40 Promotor eines zum cap-Gen heterlogen AAV Serotyps umfassen. Die Bezeichnungen P5, P19 und P40 werden für alle Serotypen verwendet (Xiao et al. (1999) J. Virol. 73:3994-4003; Bantel-Schaal et al. (1999) J. Virol. 73:949-47; Chiorini et al. (1999) J. Virol. 73:1309-19; Chiorini et al. (1997) J. Virol. 71:6823-33); für AAV4 wurde jedoch der P5-Promotor auch als P7-Promotor bezeichnet (Chiorini et al. (1997) J. Virol. 71:6823-33).Also preferred are helper constructs whose regulatory sequences for the cap gene comprise at least the P40 promoter, particularly preferably the P5 and the P40 promoter, in particular the P5, the P19 and the P40 promoter of an AAV serotype heterologous to the cap gene. The designations P5, P19 and P40 are used for all serotypes (Xiao et al. (1999) J. Virol. 73: 3994-4003; Bantel-Schaal et al. (1999) J. Virol. 73: 949-47; Chiorini et al. (1999) J. Virol. 73: 1309-19; Chiorini et al. (1997) J. Virol. 71: 6823-33); for AAV4, however, the P5 promoter was also referred to as the P7 promoter (Chiorini et al. (1997) J. Virol. 71: 6823-33).
Überraschenderweise wurde im Rahmen dieser Erfindung gefunden, dass ein cap- Gen, das allein unter der Kontrolle des homologen P40-Promotors steht, im wesentlichen konstitutiv exprimiert wird. Dies widerspricht der allgemeinen Lehrmeinung, dass für AAV2 der P40-Promotor (bzw. die entsprechenden Promotoren der anderen AAV-Serotypen) mehr oder minder allein die Expression des cap- Gens steuert (Snyder RO (1999) J. Gene Med. 1:166-75). Derartige Helferkonstrukte eignen sich somit nicht für die AAV- Verpackung, da die starke, unregulierte Expression des cap-Gens zum einen zur Bildung von überwiegend leeren Ka siden ohne AAV-Genom führt und zum anderen sich derartige Helferkon- strukte schlecht in ein zelluläres Genom integrieren lassen, da die konstitutive Expression des cap-Gens toxisch zu sein scheint. Somit ist für Cap-Helfer- konstrukte allgemein und unabhängig von den Serotypen der cap-Gene sowie der regulatorischen Sequenzen bevorzugt, dass diese neben dem P40 Promotor einen weiteren Promotor, bevorzugt den P5 und/oder P19 Promotor enthalten, welche zusammen die Expression des cap-Gens steuern. Dieses erfindungsgemäße Konzept ist somit unabhängig von den Serotypen der cap-Gene sowie der regulatorischen Sequenzen.Surprisingly, it was found in the context of this invention that a cap gene which is solely under the control of the homologous P40 promoter is essentially constitutively expressed. This contradicts the general doctrine that for AAV2 the P40 promoter (or the corresponding promoters of the other AAV serotypes) more or less controls the expression of the cap gene alone (Snyder RO (1999) J. Gene Med. 1: 166 -75). Helper constructs of this type are therefore not suitable for AAV packaging, since the strong, unregulated expression of the cap gene on the one hand leads to the formation of predominantly empty casids without an AAV genome, and on the other hand such helper constructs poorly integrated into a cellular genome because the constitutive expression of the cap gene appears to be toxic. It is therefore preferred for cap helper constructs in general and regardless of the serotypes of the cap genes and the regulatory sequences that these contain, in addition to the P40 promoter, a further promoter, preferably the P5 and / or P19 promoter, which together express the cap -Gens control. This concept according to the invention is therefore independent of the serotypes of the cap genes and of the regulatory sequences.
Bevorzugte Helferkonstrukte können zusätzlich für mindestens ein funktionelles Rep-Protein eines AAV Serotyps kodieren, der ungleich dem Serotyp A ist und vorzugsweise vom Serotyp B ist. Wie bereits oben ausgeführt, sind die erfindungsgemäßen Helferkonstrukte derart konzipiert, dass die für die Cap-Proteine kodierende Nukleinsäure operativ mit den regulatorischen Sequenzen eines ande- ren AAV-Serotyps verbunden ist. In bevorzugten Ausführungsformen kodieren diese regulatorischen Sequenzen für mindestens ein funktionelles Rep-Protein, das somit ebenfalls von einem zum Serotyp A unterschiedlichen Serotyp stammt.Preferred helper constructs can additionally code for at least one functional Rep protein of an AAV serotype which is not identical to serotype A and is preferably of serotype B. As already explained above, the helper constructs according to the invention are designed in such a way that the nucleic acid coding for the cap proteins is operatively linked to the regulatory sequences of another AAV serotype. In preferred embodiments, these regulatory sequences code for at least one functional Rep protein, which thus likewise originates from a serotype different from serotype A.
Die Rep-Proteine allgemein können aber auch durch Sequenzen kodiert werden, die im Helferkonstrukt keine regulatorischen Sequenzen der cap-Gene sind.The Rep proteins in general can also be encoded by sequences that are not regulatory sequences of the cap genes in the helper construct.
In einer bevorzugten Ausführungsform sind die Rep-Proteine Rep 68, Rep 52 und/oder Rep 40, nicht aber Rep 78, weil überraschenderweise festgestellt werden konnte, dass neben Rep 52, Rep 40 sowie den drei Cap-Proteinen VP1, VP2 und VP3 die zusätzliche Expression nur des Rep 68 für die Verpackung von AAV- Vektoren ausreichend ist. Der Vorteil dieser Rep 78-defιzienten Helferkonstrukte liegt darin, dass das größte Rep-Protein, das für die Verpackungszellen am meisten toxisch ist, überhaupt nicht exprimiert wird. Ferner wurde herausgefunden, dass Rep 78 unter den Rep- Proteinen die größte hemmende Aktivität auf zelluläre Abläufe wie beispielsweise die Transkription besitzt. Daher kann bei Verwendung dieses Helferkonstrukts aufgrund der Abwesenheit von Rep 78 die Verpackungseffizienz gesteigert werden. Sowohl Rep 68 als auch Rep 78 werden im natürlichen System durch den Promotor P5 exprimiert. Die Verwendung des Rep 78-defizienten Helferkonstrukts ist weiterhin vorteilhaft, weil Rep 68 im Vergleich zu Rep 78 in Adenovi- rus-infizierten Zellen den stärkeren Transaktivator der AAV-Promotoren P19 und P40 darstellt (Hörer et al. (1995) J. Virol. 69, 5485-5496; Weger et al. (1997) J. Virol. 71, 8437-8447). Daher führt die Verwendung dieses Rep 78-defizienten Helferkonstrukts zu einer gesteigerten Expression der kleineren Rep-Proteine Rep 40 und Rep 52 sowie der Capsidproteine und damit der gewünschten höheren Verpackungseffizienz. Dieses erfinderische Konzept gilt auch unabhängig von den Serotypen der cap-Gene sowie der regulatorischen Sequenzen.In a preferred embodiment, the Rep proteins are 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 additional expression of Rep 68 only 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, using this helper construct can increase packaging efficiency due to the absence of Rep 78. Both 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, in comparison to Rep 78 in Adenovirus-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 78-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. This inventive concept also applies regardless of the serotypes of the cap genes and the regulatory sequences.
Bevorzugte Ausführungsformen Rep 78-defιzienter Helferkonstrukte werden nachfolgend dargestellt:Preferred embodiments of Rep 78 deficient helper constructs are shown below:
Für die Herstellung des Rep 78-defizienten Helferkonstruktes pUC"Rep68,52,40Cap"(RBS)Δ37 (alias pUCRep68,52,40Cap(RBS)dl37) wurden die AAV Sequenzen von Nukleotid 201 bis Nukleotid 4497 einschließlich der Deletion der Intronsequenz sowie von Nukleotid 658 bis Nukleotid 4460 in das bakterielle Plasmid pUC19 kloniert, wobei die Bindestellen für das Rep-Protein in der ρUC19-Sequenz deletiert wurden (vgl. DE 19905501, Beispiel 5). Durch Anwendung dieser Strategie sind zwei rep- sowie wenigstens zwei cap-Gene mit jeweils einer eigenen Poly(A)-Sequenz für die Termination der Transkription hintereinander angeordnet. Ausgehend vom ersten Abschnitt (AAV-Sequenz Nukleo- tid 201 bis Nukleotid 4497) können die Rep-Proteine Rep 68 und Rep 40 sowie die Cap-Proteine VP2 und VP3 exprimiert werden, während ausgehend vom zweiten Abschnitt (AAV-Sequenz Nukleotid 658 bis Nukleotid 4460) die Rep- Proteine Rep 52 und Rep 40 sowie die Cap-Proteine VP1, VP2 und VP3 exprimiert werden. Insgesamt werden damit alle AAV2 Proteine mit Ausnahme von Rep 78 kodiert.For the production of the Rep 78-deficient helper construct pUC "Rep68,52,40Cap" (RBS) Δ37 (alias pUCRep68,52,40Cap (RBS) dl37) the AAV sequences from nucleotide 201 to nucleotide 4497 including the deletion of the intron sequence as well as from Nucleotide 658 to nucleotide 4460 cloned into the bacterial plasmid pUC19, the binding sites for the Rep protein in the ρUC19 sequence having been deleted (cf. DE 19905501, Example 5). By using this strategy, 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. Starting from the first section (AAV sequence nucleotide 201 to nucleotide 4497), the Rep proteins Rep 68 and Rep 40 and the cap proteins VP2 and VP3 are expressed, 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 AAV2 proteins with the exception of Rep 78 are encoded.
Die Herstellung derartiger Rep 78-defizienter Helferkonstrukte wird beispielhaft anhand von pUC"ΔRep78Cap"(RBS)Δ37 (alias PUCdlRep78Cap(RBS)dl37) dargestellt. Dafür wurden die AAV-Sequenzen (nt 201-2310; nt 658-4460 einschließ- lieh der Deletion der Intronsequenz) in das bakterielle Plasmid pUC19 kloniert (vgl DE 19905501, Beispiel 5). Dabei wurde die Bindestelle für das Rep-Protein in der pUC19-Sequenz deletiert. Auf diese Weise wurde das rep-Gen partiell dupliziert. Das so entstandene Helferkonstrukt enthält nur eine Poly(A)-Sequenz, so dass alle mRNA-Transkripte dasselbe 3 '-Ende besitzen. Ausgehend vom ersten Abschnitt (AAV-Sequenz Nukleotid 201 bis Nukleotid 2310) können die Rep- Proteine Rep 68 und Rep 40 exprimiert werden, während ausgehend vom zweiten Abschnitt (AAV-Sequenz Nukleotid 658 bis Nukleotid 4460) die Rep-Proteine Rep 52 und Rep 40 sowie die Cap-Proteine VP1, VP2 und VP3 exprimiert werden. Insgesamt werden damit auch durch dieses Vektorkonstrukt alle AAV2 Pro- teine mit Ausnahme von Rep 78 kodiert.The production of such Rep 78-deficient helper constructs is exemplified using pUC "ΔRep78Cap" (RBS) Δ37 (alias P UCdlRep78Cap (RBS) dl37). For this, the AAV sequences (nt 201-2310; nt 658-4460 including the deletion of the intron sequence) were cloned into the bacterial plasmid pUC19 (cf. DE 19905501, example 5). The binding site for the Rep protein in the pUC19 sequence was 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 AAV2 proteins with the exception of Rep 78.
Zur Herstellung eines Helferkonstruktes pUC"ΔReρ78ΔCap"( BS)Δ37 (alias pUCdlReρ78dlCaρ(RBS)dl37) zur Expression der Rep-Proteine Rep68, Rep52 und Rep40 wurden die AAV-Nukleotide 2945 bis 4046 aus dem cap-Gen (Nukle- otide 2203 bis 4410) des Helferkonstrukts pUC"ΔReρ78Cap"(RBS)Δ37 (alias ρUCdlRep78Cap(RBS)dl37) deletiert. Durch diese Deletion können keine funktioneilen Cap-Proteine mehr exprimiert werden. Ein weiterer Gegenstand der Erfindung ist ein System zur Verpackung von rAAV eines AAV Serotyps A bestehend aus mindestens einem der erfindungsgemäßen Helferkonstrukte, insbesondere aus einem der erfindungsgemäßen Helferkonstrukte, das nicht obligatorisch für Rep kodiert, und mindestens einem Helferkon- strukt kodierend für mindestens ein funktionelles Rep-Protein eines anderen AAV Serotyps, vorzugsweise des Serotyps B.To produce a helper construct pUC "ΔReρ78ΔCap" (BS) Δ37 (alias pUCdlReρ78dlCaρ (RBS) dl37) for the 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 "ΔReρ78Cap" (RBS) Δ37 (alias ρUCdlRep78Cap (RBS) dl37) was deleted. This deletion means that functional cap proteins can no longer be expressed. Another object of the invention is a system for packaging rAAV of an AAV serotype A consisting of at least one of the helper constructs according to the invention, in particular one of the helper constructs according to the invention, which does not necessarily code for Rep, and at least one helper construct coding for at least one functional rep Protein of another AAV serotype, preferably of serotype B.
Ein weiterer Gegenstand der vorliegenden Erfindung umfasst eine Wirtszelle zur Verpackung von einem rAAV eines AAV Serotyps A enthaltend mindestens eine Kopie eines erfindungsgemäßen Helferkonstrukts.The present invention furthermore relates to a host cell for packaging an rAAV of an AAV serotype A containing at least one copy of a helper construct according to the invention.
Geeigneterweise wird als Wirtszelle eine Säugetierzelle, insbesondere eine menschliche Zervixkarzinomzelle, vor allem eine HeLa-Zelle verwendet. HeLa- Zellen haben sich als besonders vorteilhaft erwiesen, weil der AAV-P5 -Promotor in HeLa-Zellen nahezu inaktiv ist und es deshalb möglich ist, in ihr Genom stabil eine Expressionskassette für das AAV-Rep-Protein unter der Kontrolle der natürlichen regulatorischen Elemente zu integrieren, so dass das Rep-Protein in diesen Zellen nicht toxisch wirkt (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).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 insert an expression cassette for the AAV-Rep protein into their genome under the control of the natural regulatory elements to be integrated 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).
In einer Ausführungsform liegen die Helferkonstrukte transient, also episomal transfiziert vor, in einer anderen stabil integriert in das Genom der Wirtszelle.In one embodiment, the helper constructs are transient, ie episomally transfected, in another they are stably integrated into the genome of the host cell.
AAV- Vektoren werden bevorzugt von einer Verpackungszelle oder einer Produktionszelle produziert. Dabei bezieht sich eine „Verpackungszelle" auf ein Zelle, die mindestens ein Helferkonstrukt, aber kein Vektorkonstrukt enthält. Gemäß der vorliegenden Erfindung bezieht sich eine „Produktionszelle" auf eine Zelle, welche sowohl mindestens ein Helferkonstrukt als auch mindestens ein Vektorkonstrukt enthält.AAV vectors are preferably produced by a packaging cell or a production cell. Here, a “packaging cell” refers to a cell that contains at least one helper construct, but no vector construct. According to the present invention, a “production cell” refers to a cell that contains both at least one helper construct and at least one vector construct.
Die vorliegende Erfindung betrifft somit auch eine Verpackungs- oder eine Produktionszelle, bei denen die/das Helferkonstrukt(e) in das zelluläre Genom integriert vorliegen. Dabei können mehrere gleiche oder verschiedene Helferkonstrukte an derselben Stelle, beispielsweise als Konkatemere, oder an verschiedenen Stellen integriert vorliegen.The present invention thus also relates to a packaging or a production cell in which the helper construct (s) are integrated into the cellular genome. In this case, several identical or different helper constructs can be present at the same location, for example as concatems, or integrated at different locations.
Eine solche Verpackungs- oder Produktionszelle kann von einem Helfervirus abhängig sein, wenn die AAV-Produktion eine Infektion mit einem Helfervirus erfordert. Eine solche Verpackungs- oder Produktionszelle kann aber auch Helfervirus-unabhängig sein, wenn die AAV-Produktion keine Infektion mit einem Hel- fervirus erfordert. Eine solche von einem Helfervirus unabhängige Verpackungsoder Herstellungszelle enthält normalerweise Gene, die für die Induktion der AAV-Herstellung unter der Kontrolle eines induzierbaren Promotors notwendig sind. Solche Gene können viraler oder zellulärer Herkunft sein. Unter Helfergenen werden dabei die Gene der Helferviren von AAV und/oder zelluläre Gene ver- standen, deren Genprodukte für die Replikation der AAV notwendig sind bzw. diese fördern. Beispiele für adenovirale Helfergene sind beispielsweise die Gene E1A, E1B, E4, E2A und VA E1A ist dabei für die Transaktivierung des AAV p5 Promotors erforderlich. Die Genprodukte E1B und E4 dienen hierbei zur Verstärkung der AAV mRNA- Akkumulation. Die Genprodukte E2A und VA dienen der Verstärkung des AAV mRNA-Spleißens sowie der Translation. Ferner sind als Helfergene erfindungsgemäß Herpes Simplex Virus (HSV) Helfergene eingeschlossen. Gemäß einer bevorzugten Ausführungsform sind dies die 7 Replikati- onsgene UL5, UL8, UL9, UL29, UL30, UL42 und UL52. UL 5, 8 und 52 bilden den HSV Helikase-Primase Komplex, UL29 kodiert für das Einzelstrang-DNA Bindungsprotein, UL42 für ein Doppelstrang-DNA Bindungsprotein, UL30 ko- diert für die HSV DNA Polymerase und UL9 kodiert schließlich für ein Protein, welches den HSV Replikationsursprung bindet (siehe Weindler FW and Heilbronn R (1991) J Virol. 65.(5):2476-83). Die Verwendung des Helfervirus an Stelle der einzelnen Helfergene, beispielsweise des Adenovirus-Typ 5 (Ad5), ist besonders vorteilhaft, weil dies der natürlichen Situation der AAV- Vermehrung in Gegenwart von Helferviren am nächsten kommt und somit die Verpackung von rAAV-Partikeln sehr effizient ist. Andere Helferviren sind beispielsweise Heφes- viren oder Vacciniaviren.Such a packaging or production cell may be dependent on a helper virus if AAV production requires infection with a helper virus. Such a packaging or production cell can, however, also be independent of the helper virus if the AAV production does not require infection with a helper virus. Such a packaging or manufacturing cell, which is independent of a helper virus, normally contains genes which are necessary for the induction of AAV production under the control of an inducible promoter. Such genes can be of viral or cellular origin. 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. Examples of adenoviral helper genes are, for example, the genes E1A, E1B, E4, E2A and VA. E1A 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 co dated for the HSV DNA polymerase and UL9 finally codes for a protein which binds the HSV origin of replication (see Weindler FW and Heilbronn R (1991) J Virol. 65. (5): 2476-83). The use of the 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. Other helper viruses are, for example, Heφes viruses or Vaccinia viruses.
Die Erfindung betrifft ferner die Verwendung der erfindungsgemäßen Helferkonstrukte und Zellen zur Herstellung von rAAV-Partikeln. Dabei werden geeignete Wirtszellen (s.o.) mit den erfindungsgemäßen Helferkonstrukten stabil oder tran- sient transfiziert. Anschließend oder vorher wird die Wirtszelle mit einem geeigneten Vektorkonstrukt (s.o.) stabil oder transient transfiziert. Danach wird die Zelle unter geeigneten Bedingungen kultiviert, so dass rAAV-Partikel hergestellt werden. Verfahren zur Herstellung von rAAV sind im Stand der Technik bekannt (Hauswith WW et al. (2000) Methode in Enzymology 316, 743-761; Mathews LC et al. (2002) Methods in Enzymology 346, 393-413; Potter M et al. (2002) Me- thods in Enzymology 346, 413-430; Zolotukhin S et al. (1999) Gene Therapy 6, 973-985; WO 00/47757, MediGene AG; WO 02/20748, MediGene AG).The invention further relates to the use of the helper constructs and cells according to the invention for the production of rAAV particles. Suitable host cells (see above) are transfected stably or transiently with the helper constructs according to the invention. Subsequently or beforehand, the host cell is transfected stably or transiently with a suitable vector construct (see above). The cell is then cultivated under suitable conditions, so that rAAV particles are produced. Methods for producing rAAV are known in the prior art (Hauswith WW et al. (2000) method in Enzymology 316, 743-761; Mathews LC et al. (2002) Methods in Enzymology 346, 393-413; Potter M et al . (2002) Methods in Enzymology 346, 413-430; Zolotukhin S et al. (1999) Gene Therapy 6, 973-985; WO 00/47757, MediGene AG; WO 02/20748, MediGene AG).
Die Figuren und die folgenden Beispiele sollen die Erfindung näher erläutern, ohne sie zu beschränken.The figures and the following examples are intended to explain the invention in more detail without restricting it.
Beschreibung der Figuren:Description of the figures:
Fig. 1. Fehlende AAV5 VP1 -Expression. Western Blot- Analyse der Rep/Cap- Expression in HeLa-Zellen nach Kotransfektion von pUCp5Repdl37 und pUCρ5pl9p40AAV5Caρdl37 Klone 1 und 2 sowie Adenovirus(AdV)-Infektion (MOI 10). C: Kotransfektion von ρUCp5Repdl37 und pUCp5pl9ρ40Caρdl37; M: Positivkontrolle von wtAAV/AdV-infizierten HeLa-Zellen. 48 h nach Transfekti- on erfolgte die Infektion; 48 h nach Infektion wurden die Zellen abzentrifugiert, in Zelllysepuffer lysiert und im Western Blot- Verfahren analysiert. Zur Detektion der Rep-Proteine wurde der monoklonale Antiköφer 303.9 verwendet. Die Kap- sidproteine wurden mit dem monoklonalen Antiköφer B 1 detektiert.Fig. 1. Missing AAV5 VP1 expression. Western blot analysis of Rep / Cap expression in HeLa cells after co-transfection of pUCp5Repdl37 and pUCρ5pl9p40AAV5Caρdl37 clones 1 and 2 and adenovirus (AdV) infection (MOI 10). C: co-transfection of ρUCp5Repdl37 and pUCp5pl9ρ40Caρdl37; M: Positive control of wtAAV / AdV-infected HeLa cells. The infection occurred 48 h after transfection; 48 hours after infection, the cells were centrifuged off, lysed in cell lysis buffer and analyzed in the Western blot method. The monoclonal antibody 303.9 was used to detect the Rep proteins. The capsid proteins were detected with the monoclonal antibody B 1.
Fig. 2 Korrekte AAV4 und AAV5 Kapsidprotein-Expression. Western Blot- Analyse der Kapsidprotein-Expression nach Kotransfektion von pUCp5pl9p40AAV4Cap (4Cap) bzw. pUCp5pl9p40AAV5Cap (5Cap) und pBluAAV4dlRep78dlCap (4Rep) oder PUCdlRep78dlCapdl37 (2Rep) und Infektion mit Adenovirus (MOI 10). 48 h nach Transfektion erfolgte die Infektion; 48 h nach Infektion wurden die Zellen abzentrifugiert, in Zelllysepuffer lysiert und im Western Blot- Verfahren analysiert. Zur Detektion der Kapsidproteine wurde ein Cap-spezifisches Kaninchenserum verwendet.Figure 2 Correct AAV4 and AAV5 capsid protein expression. Western blot analysis of capsid protein expression after co-transfection of pUCp5pl9p40AAV4Cap (4Cap) or pUCp5pl9p40AAV5Cap (5Cap) and pBluAAV4dlRep78dlCap (4Rep) or P UCdlRep78dlCapdl37 (MO) and I infection (10Rep) Infection occurred 48 h after transfection; 48 hours after infection, the cells were centrifuged off, lysed in cell lysis buffer and analyzed in the Western blot method. A cap-specific rabbit serum was used to detect the capsid proteins.
Fig. 3 zeigt eine schematische Darstellung des Plasmids pUCp5Repdl37.3 shows a schematic representation of the plasmid pUCp5Repdl37.
Fig. 4 zeigt eine schematische Darstellung des Plasmids pUCdlRep78- dlCap(RBS)dl37.4 shows a schematic representation of the plasmid pUCdlRep78-dlCap (RBS) dl37.
Fig. 5 zeigt eine schematische Darstellung des Plasmids pUCp5pl9p40Capdl37.5 shows a schematic representation of the plasmid pUCp5pl9p40Capdl37.
Fig. 6 zeigt eine schematische Darstellung des Plasmids pBluAAV4dlCap. Fig. 7 zeigt eine schematische Darstellung des Plasmids pBluAAV4dl- Rep78dlCap.6 shows a schematic representation of the plasmid pBluAAV4dlCap. 7 shows a schematic representation of the plasmid pBluAAV4dl-Rep78dlCap.
Fig. 8 zeigt eine schematische Darstellung des Plasmids pUCp5pl9p40AAV4Cap.8 shows a schematic representation of the plasmid pUCp5pl9p40AAV4Cap.
Fig. 9 zeigt eine schematische Darstellung des Plasmids pUCp5pl9p40AAV5Cap9 shows a schematic representation of the plasmid pUCp5pl9p40AAV5Cap
Fig. 10 zeigt einen Vergleich der Intronsequenzen einschließlich der Spleißdonor- und Spleißakzeptorstellen aller bekannten AAV-Serotypen AAVl -6. Die angege- bene Nummerierung beginnt für jeden Serotyp mit Beginn der gelisteten Sequenz mit 1. Das schwarze Dreieck zeigt die Schnittstelle der Spleißdonorstelle an, die weißen Dreiecke die der Spleißakzeptorstellen.10 shows a comparison of the intron sequences including the splice donor and splice acceptor sites of all known AAV serotypes AAVl -6. The numbering given for each serotype begins with the beginning of the listed sequence with 1. The black triangle shows the interface of the splice donor site, the white triangles that of the splice acceptor sites.
Fig. 11 zeigt eine schematische Darstellung von Trans-split Helferkonstrukten sowie Cis-split Helferkonstrukten.11 shows a schematic representation of trans-split helper constructs and cis-split helper constructs.
BeispieleExamples
Im Rahmen dieser Erfindung wurden cap-Helferplasmide für verschiedene Serotypen, hier AAV4 und AAV5, hergestellt, die in Kombination mit einem AAV2 Rep-Helferplasmid, einem rekombinanten AAV2 Vektorplasmid und bei Infektion mit einem Helfervirus hybrides rAAV ergeben. Dieses System ist zum einen sehr flexibel, da hierbei lediglich das cap-Helferplasmid ausgetauscht werden muss, um hybrides rAAV mit einem Kapsid eines anderen AAV-Serotyps zu erhalten. Zum anderen ist auch hier die Entstehung von replikationskompetentem AAV, der durch die Verwendung von rep und cap verschiedener Serotypen in diesem Fall sogar einen komplett neuen „Wildtyp"-AAV darstellte - mit bisher nicht absehbaren Folgen im Patienten -, durch die Notwendigkeit von zwei Rekombinationsereignissen weitestgehend ausgeschlossen.In the context of this invention, cap helper plasmids for various serotypes, here AAV4 and AAV5, were produced, which in combination with an AAV2 Rep helper plasmid, a recombinant AAV2 vector plasmid and, when infected with a helper virus, gave hybrid rAAV. On the one hand, this system is very flexible, since only the cap helper plasmid has to be exchanged in order to obtain hybrid rAAV with a capsid of another AAV serotype. On the other hand, the development of replication-competent AAV, which is achieved through the use of rep and cap of different serotypes This case even represented a completely new "wild-type" AVA - with previously unpredictable consequences in the patient - largely excluded due to the need for two recombination events.
Zusammengefasst wurde der offene Leserahmen von AAV4 cap bzw. AAV5 cap unter die Kontrolle des AAV2 p40-Promotors (sowie AAV2 p5 und pl9) kloniert, um mögliche Inkompatibilitäten zwischen Promotoren oder Rep-Proteinen und Promotoren verschiedener AAV-Serotypen auszuschließen. Überraschenderweise wurde festgestellt, dass die Verwendung der rein kodierenden cap-Sequenzen zur Bildung nicht-infektiöser Partikel führte, was darauf zurückgeführt werden konnte, dass zwar die Kapsidproteine VP2 und VP3 von AAV4 bzw. AAV5 gebildet wurden, nicht aber das entsprechende VP1 (siehe Fig. 1). In der Literatur war bereits beschrieben worden, dass die Kapsidproteine VP2 und VP3 von AAV2 allein zur Partikelbildung führen, diese aber ohne VP1 nicht infektiös sind (Smuda und Carter 1991, Virology 184:310-8).In summary, the open reading frame of AAV4 cap or AAV5 cap was cloned under the control of the AAV2 p40 promoter (as well as AAV2 p5 and pl9) in order to rule out possible incompatibilities between promoters or Rep proteins and promoters of different AAV serotypes. Surprisingly, it was found that the use of the purely coding cap sequences led to the formation of non-infectious particles, which could be attributed to the fact that the capsid proteins VP2 and VP3 were formed by AAV4 and AAV5, but not the corresponding VP1 (see FIG . 1). It has already been described in the literature that the capsid proteins VP2 and VP3 of AAV2 alone lead to particle formation, but without VP1 they are not infectious (Smuda and Carter 1991, Virology 184: 310-8).
Es wurde nun überraschenderweise gefunden, dass ein verändertes Spleißverhalten aufgrund des Vorhandenseins des Introns von unterschiedlichen AAV- Serotypen vorlag. Dies konnte anhand von neuen AAV4 bzw. AAV5 Cap- Helfeφlasmiden gezeigt werden, die zusätzlich zu dem offenen AAV4 bzw. AAV5 cap-Leserahmen auch die gesamte Intronsequenz inklusive der Spleißdo- norstelle von AAV4 bzw. AAV5 enthalten.It has now surprisingly been found that there was a change in splicing behavior due to the presence of the intron of different AAV serotypes. This could be demonstrated using new AAV4 or AAV5 cap helper phasmids, which in addition to the open AAV4 or AAV5 cap reading frame also contain the entire intron sequence including the splice donor site of AAV4 and AAV5.
1. Herstellung der Vektoren1. Production of the vectors
Das AAV2 rep-Helferplasmid P5Rep wurde durch Deletion eines DNA- Fragments hergestellt, das die Nukleotide 2300-4170 des AAV-Genoms enthielt. (Ruffing et al. (1994) J. Gen. Virol. 75, 3385-3392 (Gene Bank Accession No. AF 043303). P5RepΔ37 (alias pUCρ5Reρdl37) wurde durch Deletion der AAV- Basen 4461 - 4497 aus P5Rep gewonnen (Fig. 3).The AAV2 rep helper plasmid P5Rep was prepared by deleting a DNA fragment that contained nucleotides 2300-4170 of the AAV genome. (Ruffing et al. (1994) J. Gen. Virol. 75, 3385-3392 (Gene Bank Accession No. AF 043303). P5RepΔ37 (alias pUCρ5Reρdl37) was obtained from P5Rep by deleting the AAV bases 4461-4497 (FIG. 3).
Das AAV2 rep-Helferkonstrukt ρUC"ΔReρ78ΔCaρ"(RBS)Δ37 (alias ρUCΔReρ78ΔCap(RBS)Δ37 oder pUCdlRep78dlCaρdl37) wurde durch Deletion der Nukleotide 3046 bis 4149 aus dem Helferkonstrukt pUC"ΔRep78Cap"- (RBS)Δ37 (alias pUCdlRep78Cap(RBS)dl37; Klonierung siehe WO 00/47757 S.26 Z.14 bis S.29 Z.5) unter Verwendung des Restriktionsenzyms Apal hergestellt (Fig 4).The AAV2 rep helper construct ρUC "ΔReρ78ΔCaρ" (RBS) Δ37 (alias ρUCΔReρ78ΔCap (RBS) Δ37 or pUCdlRep78dlCaρdl37) was obtained by deleting the nucleotides 3046 to 4149 from the helper construct pUC "ΔRep78Capd ; Cloning see WO 00/47757 p.26 Z.14 to S.29 Z.5) prepared using the restriction enzyme Apal (Fig. 4).
Das AAV2 cap-Helferplasmid P5P19P40Caρ (alias pUCρ5pl9p40Caρ) wurde wie folgt hergestellt: zunächst wurde das cap-Gen mit BamHI-SnaBI aus AAV2 herausgeschnitten und in die BamHI-Smal-Restriktionsstellen von pUC19 kloniert, um das Plasmid pUCcap zu generieren. Anschließend wurden die Nukleoti- de 200-1056 des AAV2 rep-Gens mittels PCR amplifiert und über Xbal-BamHI in pUCcap inseriert, um das AAV2 rep/cap Helferplasmid pUC"rep/cap" herzustellen. Daraufhin wurde das AAV2 Rep 78- und Rep 68-defiziente Helferplasmid pUCρ5pl9Rep52(40)p40Cap durch Deletion der Nukleotide 6522-6828 von pUC"rep/cap" mittels PpuMI-NruI-Nerdau generiert. Durch eine weitere Deletion der Nukleotide 5620- 6277 von pUCp5pl9Rep52(40)p40Cap mittels BamHI- BstEII-Verdau und Blunting der Fragmentenden entstand das AAV2 cap- Helferplasmid pUCp5pl9p40Cap.The AAV2 cap helper plasmid P5P19P40Caρ (alias pUCρ5pl9p40Caρ) was produced as follows: first, the cap gene was cut out of AAV2 with BamHI-SnaBI and cloned into the BamHI-Smal restriction sites of pUC19 in order to generate the plasmid pUCcap. The nucleotides 200-1056 of the AAV2 rep gene were then amplified by PCR and inserted into pUCcap via Xbal-BamHI in order to produce the AAV2 rep / cap helper plasmid pUC "rep / cap". The AAV2 Rep 78 and Rep 68 deficient helper plasmid pUCρ5pl9Rep52 (40) p40Cap was then generated by deleting nucleotides 6522-6828 from pUC "rep / cap" using PpuMI-NruI-Nerdau. The AAV2 cap helper plasmid pUCp5pl9p40Cap resulted from a further deletion of nucleotides 5620-6277 from pUCp5pl9Rep52 (40) p40Cap by means of BamHI-BstEII digestion and blunting of the fragment ends.
Das AAV2 rep/cap-Helferplasmid pUC"rep/cap"(RBS)Δ37 wurde wie folgt gene- riert: zunächst wurden die Nukleotide 267-291 von pUC"rep/cap" (s.o.) mittels PCR-Mutagenese deletiert, um das Plasmid pUCAAVSapI zu erzeugen. Anschließend wurden die Nukleotide 2660-2696 von pUCAAVSapI mittels PCR- Mutagenese deletiert, um das AAV2 rep/cap-Helfeφlasmid pUC"rep/cap"- (RBS)Δ37 herzustellen. Das AAV2 cap-Helferplasmid pUCp5pl9p40Capdl37 (alias pUCp5ρl9p40- CapΔ37) wurde über den Austausch der Nukleotide 4459 - 2989 (Restriktionsenzyme BsiWI und Ndel) von pUCp5pl9p40Cap durch nt 2426-3857 (Ndel-BsiWI) von ρUC"rep/caρ"(RBS)Δ37 hergestellt (Fig. 5).The AAV2 rep / cap helper plasmid pUC "rep / cap" (RBS) Δ37 was generated as follows: First, the nucleotides 267-291 of pUC "rep / cap" (see above) were deleted by means of PCR mutagenesis to remove the plasmid to generate pUCAAVSapI. The nucleotides 2660-2696 of pUCAAVSapI were then deleted by means of PCR mutagenesis in order to produce the AAV2 rep / cap-Helfeφlasmid pUC "rep / cap" - (RBS) Δ37. The AAV2 cap helper plasmid pUCp5pl9p40Capdl37 (alias pUCp5ρl9p40-CapΔ37) was obtained via the exchange of nucleotides 4459-2989 (restriction enzymes BsiWI and Ndel) from pUCp5pl9p40Cap by nt 2426-3857 (Ndel-BsiUC ") from ΔiBi" Rep made (Fig. 5).
Das AAV4 rep-Helferplasmid pBluAAV4dlCap wurde durch die Deletion von nt 3262-4407 (Sful-Smal) von ρAAV4-2 (bezogen von NIH; siehe Chiorini et al. (1997) J. Virol. 71:6823-33) hergestellt (Fig. 6).The AAV4 rep helper plasmid pBluAAV4dlCap was prepared by deleting nt 3262-4407 (Sful-Smal) from ρAAV4-2 (obtained from NIH; see Chiorini et al. (1997) J. Virol. 71: 6823-33) (Fig 6).
Für die Herstellung des Rep78-defizienten AAV4 rep-Helferplasmids pBlu- AAV4dlRep78dlCap wurde zunächst das Plasmid pBluAAV4Rep68,40Cap durch Deletion von nt 2445-2770 von pAAV4-2 über PCR-basierte Mutagenese hergestellt. Anschließend wurden die Nukleotide 3906-4498 (Xbal-Mlul) von pBlu- AAV4dlCap durch nt 677-2937 (Xbal-Sfol) von pBluAAV4Rep68,40Cap ausgetauscht (Fig. 7).For the production of the Rep78-deficient AAV4 rep helper plasmid pBlu-AAV4dlRep78dlCap, the plasmid pBluAAV4Rep68,40Cap was first prepared by deleting nt 2445-2770 from pAAV4-2 via PCR-based mutagenesis. The nucleotides 3906-4498 (Xbal-Mlul) from pBlu-AAV4dlCap were then replaced by nt 677-2937 (Xbal-Sfol) from pBluAAV4Rep68.40Cap (FIG. 7).
Für die Herstellung des AAV4 cap-Helferplasmids pUCp5pl9p40AAV4Cap wurde zunächst durch PCR-basierte Mutagenese eine Notl-Schnittstelle an die Position nt 3156 von pUCp5pl9p40Capdl37 eingefügt, um das Zwischenprodukt pUCp5pl9p40Capdl37Not2 zu generieren. Anschließend wurden die Nukleotide 348-3156 (Ndel-Notl) von pUCρ5pl9p40Caρdl37Not2 durch die mittels PCR amplifizierten nt 2365-5164 von ρAAV4-2 über Notl-Ndel ersetzt (Fig. 8). (Für die Herstellung des entsprechenden Konstrukts pUCp5pl9p40AAV4Capdl37 oh- ne die AAV4 Spleißdonorstelle war bei sonst identischer Klonierungsstrategie ein vom 5 '-Ende her um ca. 100 nt verkürztes Fragment amplifiziert worden.) Das AAV5 cap-Helferplasmid pUCp5pl9p40AAV5Cap wurde über den Austausch der Nukleotide 348-3156 (Ndel-Notl) von pUCp5pl9ρ40Caρdl37Not2 durch die mittels PCR amplifierten nt 2305-5095 von pAAV5-2 (bezogen von NIH; siehe Chiorini et al. (1999) J. Virol. 73:1309-19) über Notl-Ndel ersetzt (Fig. 9). (Für die Herstellung des entsprechenden Konstrukts pUCp5pl9p40- AAV5Capdl37 ohne die AAV4 Spleißdonorstelle war bei sonst identischer Klonierungsstrategie ein vom 5 '-Ende her um ca. 100 nt verkürztes Fragment ampli- Fiziert worden.)For the production of the AAV4 cap helper plasmid pUCp5pl9p40AAV4Cap, a NotI interface was first inserted at position nt 3156 of pUCp5pl9p40Capdl37 by PCR-based mutagenesis in order to generate the intermediate product pUCp5pl9p40Capdl37Not2. The nucleotides 348-3156 (Ndel-Notl) of pUCρ5pl9p40Caρdl37Not2 were then replaced by the PCR-amplified nt 2365-5164 from ρAAV4-2 via Notl-Ndel (FIG. 8). (For the production of the corresponding construct pUCp5pl9p40AAV4Capdl37 without the AAV4 splice donor site, a fragment shortened by about 100 nt from the 5 'end was amplified with an otherwise identical cloning strategy.) The AAV5 cap helper plasmid pUCp5pl9p40AAV5Cap was obtained by replacing the nucleotides 348-3156 (Ndel-Notl) of pUCp5pl9ρ40Caρdl37Not2 with the PCR-amplified nt 2305-5095 from pAAV5-2 (obtained from NIH; see Chiorini J. et al. (1999) Virol. 73: 1309-19) via Notl-Ndel (Fig. 9). (For the production of the corresponding construct pUCp5pl9p40-AAV5Capdl37 without the AAV4 splice donor site, a fragment shortened by about 100 nt from the 5 'end was amplified with an otherwise identical cloning strategy.)
2. Verpackung von hybriden rAAV mit unterschiedlichen Helferkonstrukten mit Spleißdonor- und Spleißakzeptorstelle abgeleitet von unterschiedlichen Serotypen2. Packaging of hybrid rAAV with different helper constructs with splice donor and splice acceptor site derived from different serotypes
Hybride rAAV wurden in HeLa-Zellen nach Kotransfektion von pUCp5Repdl37 und pUCρ5pl9p40AAV5Caρdl37 Klone 1 und 2 sowie Adenovirus(AdV)- Infektion (MOI 10) nach Standardmethoden hergestellt (siehe z.B. WO 00/47757). Bei diesem cap-Helferkonstrukt befinden sich die kodierenden Sequenzen des cap-Gens von AAV5 unter der Kontrolle der regulatorischen Sequenzen von AAV2, wobei diese AAV2-Sequenzen die AAV2 Cap-Spleiß- donorstelle umfasst. Somit sind Spleißdonor- und Spleißakzeptorstelle von unterschiedlichen Serotypen, AAV2 und AAV5, abgeleitet.Hybrid rAAV were produced in HeLa cells after co-transfection of pUCp5Repdl37 and pUCρ5pl9p40AAV5Caρdl37 clones 1 and 2 as well as adenovirus (AdV) infection (MOI 10) according to standard methods (see e.g. WO 00/47757). In this cap helper construct, the coding sequences of the AAV5 cap gene are under the control of the regulatory sequences of AAV2, these AAV2 sequences comprising the AAV2 cap splice donor site. Thus, splice donor and splice acceptor sites are derived from different serotypes, AAV2 and AAV5.
Als Kontrolle dienten eine Kotransfektion von pUCp5Repdl37 und pUCρ5pl9p40Capdl37 (C) sowie wtAAV/AdV-infizierten HeLa-Zellen (M).A cotransfection of pUCp5Repdl37 and pUCρ5pl9p40Capdl37 (C) and wtAAV / AdV-infected HeLa cells (M) served as controls.
48 h nach Transfektion erfolgte die Infektion; 48 h nach Infektion wurden die Zellen abzentrifugiert, in Zelllysepuffer lysiert und im Westerablot- Verfahren analysiert. Zur Detektion der Rep-Proteine wurde der monoklonale Antikörper 303.9 (Progen, Heidelberg, Deutschland) verwendet. Die Kapsidproteine wurden mit dem monoklonalen Antikörper Bl detektiert (Wistuba et al. (1997) J. Virol. 71, 1341-1353).Infection occurred 48 h after transfection; 48 hours after infection, the cells were centrifuged off, lysed in cell lysis buffer and analyzed using the Westerablot method. To detect the Rep proteins, the monoclonal antibody 303.9 (Progen, Heidelberg, Germany) used. The capsid proteins were detected with the monoclonal antibody B1 (Wistuba et al. (1997) J. Virol. 71, 1341-1353).
Fig. 1 zeigt in einer Westernblotanalyse, dass für beide Klone 1 und 2 des Verpackungsexperiments im Gegensatz zu den Kontrollen M und C keine Expression von VP1 erkennbar ist (siehe auch Beschreibung der Fig. 1).1 shows in a Western blot analysis that for both clones 1 and 2 of the packaging experiment, in contrast to controls M and C, no expression of VP1 can be seen (see also description of FIG. 1).
3. Verpackung von hybriden rAAV mit unterschiedlichen Helferkonstruk- ten mit Spleißdonor- und Spleißakzeptorstelle abgeleitet von gleichen Serotypen3. Packaging of hybrid rAAV with different helper constructs with splice donor and splice acceptor sites derived from the same serotypes
Hybride rAAV wurden in HeLa-Zellen nach Kotransfektion von pUCp5pl9p40AAV4Cap (4Cap) bzw. pUCp5pl9p40AAV5Cap (5Cap) und pBluAAV4dlRep78dlCap (4Rep) oder pUCdlRep78dlCapdl37 (2Rep) und Infektion mit Adenovirus (MOI 10) hergestellt.Hybrid rAAV were produced in HeLa cells after cotransfection of pUCp5pl9p40AAV4Cap (4Cap) or pUCp5pl9p40AAV5Cap (5Cap) and pBluAAV4dlRep78dlCap (4Rep) or pUCdlRep78dlCapdl37 (MOI10D) and infection (MOI 10Dep).
48 h nach Transfektion erfolgte die Infektion; 48 h nach Infektion wurden die Zellen abzentrifugiert, in Zelllysepuffer lysiert und im Western Blot- Verfahren ana- lysiert. Zur Detektion der Kapsidproteine wurde ein Cap-spezifisches Kaninchenserum verwendet.Infection occurred 48 h after transfection; 48 hours after infection, the cells were centrifuged, lysed in cell lysis buffer and analyzed using the Western blot method. A cap-specific rabbit serum was used to detect the capsid proteins.
Fig. 2 zeigt in einer Westernblotanalyse, dass in allen getesteten Kombinationen von cap-Genen (für AAV4 (4Cap) und AAV5 (5Cap)) mit den regulatorischen Sequenzen von AAV4 (4Rep) und AAV2 (2Rep) zur korrekten AAN4 und AAV5 Kapsidprotein-Expression führten. 2 shows in a Western blot analysis that in all tested combinations of cap genes (for AAV4 (4Cap) and AAV5 (5Cap)) with the regulatory sequences of AAV4 (4Rep) and AAV2 (2Rep) for correct AAN4 and AAV5 capsid protein Expression resulted.

Claims

Patentansprüche claims
1. Helferkonstrukt zur Verpackung von rAAV, bei dem1. Helper construct for packaging rAAV, in which
a) die für die Cap-Proteine eines Serotyps A kodierenden Nukleinsäuren operativ mit regulatorischen Sequenzen eines zweiten AAV Serotyps B verbunden sind, b) das Intron der für die Cap-Proteine kodierenden Nukleinsäuren vom Serotyp A ist, und c) die für die Cap- und Rep-Proteine kodierenden Nukleinsäuren funktioneil getrennt vorliegen.a) the nucleic acids coding for the cap proteins of a serotype A are operatively linked to regulatory sequences of a second AAV serotype B, b) the intron of the nucleic acids coding for the cap proteins is of serotype A, and c) that for the cap proteins and nucleic acids encoding Rep proteins are functionally separate.
2. Helferkonstrukt nach Anspruch 1, dadurch gekennzeichnet, dass das Intron die Spleißdonor- und Spleißakzeptorstellen einschließt.2. Helper construct according to claim 1, characterized in that the intron includes the splice donor and splice acceptor sites.
3. Helferkonstrukt nach den Ansprüchen 1 oder 2, dadurch gekennzeichnet, dass die regulatorischen Sequenzen mindestens den P40 Promotor, bevorzugt den P5 und den P40 Promotor, besonders bevorzugt den P5, den P19 und den P40 Promotor umfassen.3. Helper construct according to claims 1 or 2, characterized in that the regulatory sequences comprise at least the P40 promoter, preferably the P5 and the P40 promoter, particularly preferably the P5, the P19 and the P40 promoter.
4. Helferkonstrukt nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die Serotypen A und B ausgewählt sind aus den Serotypen AAVl, AAV2, AAV3, AAV4, AAV5 und AAV6.4. Helper construct according to one of claims 1 to 3, characterized in that the serotypes A and B are selected from the serotypes AAVl, AAV2, AAV3, AAV4, AAV5 and AAV6.
5. Helferkonstrukt nach Anspruch 4, dadurch gekennzeichnet, dass der Serotyp B AAV2 ist. 5. helper construct according to claim 4, characterized in that the serotype B is AAV2.
6. Helferkonstrukt nach einem der Ansprüche 4 oder 5, dadurch gekennzeichnet, dass der Serotyp A AAV4 oder AAV5 ist.6. helper construct according to one of claims 4 or 5, characterized in that the serotype A is AAV4 or AAV5.
7. Helferkonstrukt nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass das Konstrukt zusätzlich für mindestens ein funktionelles Rep-Protein eines AAV Serotyps kodiert, der nicht Serotyp A ist, vorzugsweise eines Serotyps B.7. helper construct according to one of claims 1 to 6, characterized in that the construct additionally codes for at least one functional Rep protein of an AAV serotype that is not serotype A, preferably a serotype B.
8. System zur Verpackung von einem rAAV bestehend aus a) mindestens einem Helferkonstrukt nach den Ansprüchen 1 bis 7, oder b) aus mindestens einem Helferkonstrukt nach einem der Ansprüche 1 bis 6 und mindestens einem Helferkonstrukt kodierend für mindestens ein funktionelles Rep-Protein eines Serotyps, der nicht Serotyp A ist, vor- zugsweise eines Serotyps B.8. System for packaging a rAAV consisting of a) at least one helper construct according to claims 1 to 7, or b) from at least one helper construct according to one of claims 1 to 6 and at least one helper construct coding for at least one functional Rep protein of a serotype that is not serotype A, preferably a serotype B.
9. Wirtszelle zur Verpackung von einem rAAV eines AAV Serotyps A enthaltend mindestens eine Kopie eines Helferkonstrukts nach einem der Ansprüche 1 bis 6.9. host cell for packaging an rAAV of an AAV serotype A containing at least one copy of a helper construct according to any one of claims 1 to 6.
10. Verwendung der Helferkonstrukte nach den Ansprüchen 1-7 oder des Systems zur Verpackung von einem rAAV gemäß Anspruch 8 oder einer Wirtszelle gemäß Anspruch 9 zur Herstellung von rAAV Partikeln. 10. Use of the helper constructs according to claims 1-7 or the system for packaging an rAAV according to claim 8 or a host cell according to claim 9 for the production of rAAV particles.
PCT/EP2003/002351 2002-03-07 2003-03-07 HELPER CONSTRUCTS FOR PRODUCING HYBRID rAAV PARTICLES OF VARIOUS AAV SEROTYPES WO2003074686A1 (en)

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