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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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

Definitions

• the invention relates to the field of genetically engineered viral vectors, more specifically to adeno- associated virus (AAV) vectors, for use in gene therapy.
• Adeno-associated virus is a non-pathogenic human parvovirus (reviewed in (Berns, 1990a; Berns, 1990b)). The virus replicates as a single strand DNA of approximately 4.6 kb . Both the plus and the minus strand are packaged and infectious. Efficient replication of AAV requires the co- infection of the cell by a helper virus .
• Viruses which have been identified to help AAV are the adenoviruses, herpes simplex virus (HSV) , cytomegalovirus (CMV) and pseudorabies virus (Berns, 1996) .
• HSV herpes simplex virus
• CMV cytomegalovirus
• pseudorabies virus Borabies virus
• AAV is therefore also classified as a dependovirus .
• the AAV genome can integrate into the host cell genome.
• the wild-type virus has a strong preference (70%) for an integration site on the long arm of chromosome 19 (ql3.3- qter) (Kotin et al, 1990; Samulski, 1993; Samulski et al, 1991) . Following integration, the expression of the virus genes is not detectable.
• the integrated provirus replicates as a normal part of the host cell genome upon division of the transduced cell and ends up in both daughter cells.
• This stage of the virus life cycle is known as the latent stage.
• This latent stage is stable but can be interrupted upon infection of the transduced cell by a helper virus.
• AAV is excised from the host cell genome and starts to replicate.
• the rep-genes are expressed.
• the capsid proteins VP1, VP2 en VP3 are produced in detectable amounts and the replicated virus DNA is packaged into virions .
• a schematic representation of the AAV-genome and its genes is depicted in figure 1.
• the virions accumulate in the nucleus of the cell and are released when the cell lyses as a result of the accumulation of AAV and the helper virus (reviewed in Berns, 1990a; Berns, 1990b) .
• Six primate AAV serotypes have been characterised thus far (Berns et al, 1994; Rutledge et al, 1998) .
• the AAV-genome contains two genes rep and cap (Fig. 1) .
• Three promoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4 Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins (VPl, VP2 and VP3) .
• the AAV- genome is flanked on both sides by a 145 bp sequence, called the Inverted Terminal Repeat (ITR) , which appears to contain all the cis-acting sequences required for virus integration, replication and encapsidation (Lusby et al, 1980; Samulski et al, 1989) .
• ITR Inverted Terminal Repeat
• Rep78 and Rep68 are essential for AAV-replication and trans regulation of viral genes.
• Rep52 and Rep40 are expressed from the P19 promoter and are thought to be involved in the packaging of AAV-genomes (Chejanovsky and Carter, 1989; Smith and Kotin, 1998) .
• the capsid proteins VPl, VP2 and VP3 are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNAs by using the same splice donor but two different splice acceptor sites. The splice acceptor sites are located at both sides of the VPl translation start signal.
• VPl is translated from the messenger that uses the splice acceptor directly in front of the VPl translation initiation codon .
• VP2 and VP3 are translated from the messenger RNA that is spliced to the acceptor 3' of the VPl ATG.
• the proteins VP2 and VP3 are translated from this messenger by use of an ACG translation start (VP2) or a downstream ATG (VP3). Since all three coding regions are in frame, the capsid proteins share a large domain with an identical amino-acid sequence.
• VP3 is entirely contained within VPl and VP2, but the latter two contain additional amino-terminal sequences.
• VPl contains the entire VP2 protein but carries an additional N-terminal sequence.
• the AAV capsid is 20 to 24 nm in diameter (Berns and Bohensky, 1987; Srivastava et al, 1983) and contains approximately 5% VPl, 5% VP2 and 90% VP3. This ratio is believed to reflect the relative abundance of the alternatively spliced messengers and the reduced translation initiation efficiency at the ACG initiation codon for VP2.
• Adeno-associated virus vectors can be made by replacing the rep- and cap-gene sequences in the wild type AAV with the sequence of interest.
• concomitant helper virus infected human cells need to be supplied with the rep- and cap-genes through different means. This is routinely done through transfection of a so-called packaging plasmid, providing AAV-packaging function, containing the AAV-rep- and cap-genes but lacking the AAV- ITRs .
• Recombinant AAV is typically generated by co- transfecting a packaging plasmid together with a plasmid containing the recombinant AAV into helper virus infected cells .
• the recombinant virus is typically harvested from such cultures 48 to 72 hours after transfection of the cells.
• Recombinant AAV generated in this way is high titre and can be made essentially free of wild type AAV (Allen et al, 1997; Samulski et al, 1989) . Since the cells are also concomitantly infected with a helper virus, usually adenovirus, this helper virus is also produced (Clark et al, 1997; Flotte et al,
• AAV-replication and also packaging can be accomplished in the test tube using a cell free system (Hong et al, 1992; Hong et al, 1994; ⁇ i et al, 1994; Zhou and Muzyczka, 1998; Ward et al, 1998) .
• helper virus in rAAV preparations is not desirable.
• the concomitant helper virus is a potential pathogen and even minor contaminations of recombinant AAV preparations with the helper virus are not acceptable for clinical use.
• Several methods are employed to remove the helper virus from the recombinant AAV preparations. In case of adenovirus these include differences in density and temperature sensitivity. AAV particles have a density of 1.41 to 1.45 g/cm 3 whereas adenovirus 2 and 5, the most commonly used helper viruses have a density of 1.33 g/cm 3 . With density gradient centrifugation this difference is utilised to separate the two viruses (Clark et al, 1997; Herzog et al, 1997) .
• Differences in temperature sensitivity are also used to remove contaminating adenovirus.
• Adeno-associated virus particles are more resistant to heat treatment than adenovirus particles.
• Routinely, recombinant AAV preparations are incubated for 1 hour at 56 °C .
• the recombinant AAV is resistant to this treatment whereas the adenovirus helper virus is not (Flotte et al, 1993; Monahan et al, 1998; Snyder et al, 1997a) .
• these methods are adequate to remove most of the helper virus they are not ideal for clinical applications of recombinant AAV.
• One reason is that for clinical applications large amounts of recombinant AAV need to be produced. This implies that also large amounts of helper virus are produced which must then subsequently be removed completely from the rAAV preparation.
• the process of validating the absence of the helper virus is difficult.
• the invention is directly related to the generation, production and purification of genetically engineered viral vectors designed to introduce and express a gene of interest in mammalian cells.
• the present invention provides a process for the production of high titer recombinant adeno-associated virus (AAV) vectors that are essentially free of helper virus such as adenovirus.
• AAV adeno-associated virus
• Several viruses can provide helper functions for AAV.
• the helper function of adenovirus is momentarily the best characterised.
• adenovirus four regions have been identified that are required for fully permissive AAV infection. These are the El, E2a, E4orf6 and VA regions.
• genes from both the Ela and the Elb region are important.
• HSV can also function as a helper virus for AAV.
• HSV genes with helper virus function identified so far include the ICP 8 and the IPC 4 genes, the viral DNA polymerase and possibly the viral helicase (Berns, 1996) .
• the invention provides an adeno-associated virus (AAV) packaging cell having been provided with nucleic acid encoding a gene product providing AAV helper function allowing generating recombinant AAV without concomitant helper virus production.
• AAV adeno-associated virus
• the invention provides methods, cell lines, recombinant adenoviral vectors and recombinant DNA molecules especially suited for the large scale production of high titer recombinant AAV stocks that are free of replication competent adenovirus.
• AAV replication and packaging is undetectable .
• low level replication and packaging in the absence of helper virus function can be induced.
• Several methods have been published to induce a productive replicative cycle of AAV on a low scale.
• the invention provides a structurally better solution to completely avoid the generation of helper virus during the production of recombinant AAV. Preventing the generation of the helper virus avoids the requirement for painstaking purification and subsequent validation and testing of the preparations .
• the invention provides the way to eliminate the generation of helper virus during the production of AAV by eliminating the helper virus requirement of AAV- replication .
• a packaging cell which expresses the adenovirus E2A-gene and additionally required helper functions, where the additionally required helper functions do not posses sequence overlap with the E2A-helper function already present in the packaging cell, leading to the formation of RCA.
• the E2A-gene is derived from the adenovirus tsl25.
• a packaging cell which expresses the adenovirus El-region and additionally required helper functions, where the additionally required helper functions do not posses sequence overlap with the El-helper function already present in the packaging cell, leading to the formation of RCA.
• the packaging cell comprises the PER cell lines.
• the PER cell lines have been generated from normal human embryonic retinoblast (HER) cells which were immortalised with a fully characterised plasmid containing the human adenovirus 5 El-region (WO 97/00326) .
• the PER cells are specifically useful in preventing the formation of RCA in combination with novel El-deleted adenovirus vectors (WO 97/00326) which do not posses sequence overlap with the El- region present in PER cells.
• PER cells are supplied with the additionally required helper virus function through infection of an El-deleted adenovirus that contains no sequence overlap with the El-sequences already present in the PER cells, leading to the formation of RCA.
• the PER cells are provided with the additionally required helper virus function through transfection with plasmid DNA containing the helper virus function encoding genes and which plasmid contains no sequence overlap with the El sequences already present in the PER cells, leading to the formation of RCA.
• An example of such a PER cell, PER.C6 has been deposited under accession number 96022940 ECACC at the Centre for Applied Microbiology Research (CAMR) .
• the most commonly used cell lines for the production of rAAV are HeLa and 293. Although these cell lines are widely used there are several drawbacks attached to them.
• the HeLa cells are derived from human cancer and thus carry one or more oncogenes in their DNA.
• the 293 cells have the advantage that they are not derived from human cancer. However, they are stably transfected with some adenovirus sequences and as a result of that express El-genes (Graham et al, 1977a). This El-gene expression is sufficient for the production of recombinant AAV (Herzog et al, 1997;
• the 293 cell line has a disadvantage. Not only the El-region is stably integrated into the DNA of the cells. From the left hand side of the adenovirus genome it is known that the cell line carries at least adenovirus 5 sequences 1-4344 containing the left hand ITR, the packaging signal, the El-gene and the gene encoding protein IX (Louis et al, 1997) . The presence of more than just the El-sequences leaves a significant region of overlap on both sides with the most commonly used El-deleted adenovirus vectors or deletion mutants such as dl312 (Snyder et al, 1997b) .
• the region of overlap is sufficient for homologous recombination between the most commonly used El- deleted adenovirus vectors and the adenovirus 5 sequences in 293.
• Such a homologous recombination event can lead to the undesired generation of replication competent adenovirus (RCA) (Hehir et al, 1996) .
• RCA replication competent adenovirus
• the presence of RCA in El-deleted adenovirus vector stocks is a problem (Imler et al, 1996; Lochmuller et al, 1994) .
• the invention described in (Ferrari et al, 1996) and WO 96/40240 comprises the transfection of 293 cells with a 35,000 bp DNA fragment isolated from Xbal digested DNA from adenovirus dl309 to provide for the adenovirus helper functions for the production of recombinant AAV.
• This technique is not ideal since this Xbal fragment has considerable overlap with the adenovirus sequences in 293, enabling the inadvertent generation of replication competent adenovirus.
• Another disadvantage is that dl309 has an insertion of DNA in the E3-region.
• Fine tuning of the technique has led to the generation of adenovirus helper plasmids with deletions of adenovirus genes while retaining the helper virus function for recombinant AAV production (WO 97/17458, Ferrari et al, 1997; Li et al, 1997; Xiao et al, 1998a) .
• Using these adenovirus late gene deleted helper plasmids to avoid RCA is in general restricted to 293 cells.
• this cell line has several disadvantages, one further disadvantage of the 293 cell line is it that only expresses the El-region and thus additionally requires helper function for efficient and large scale production of recombinant AAV, which need to be supplied separately. Furthermore, the culturing of 293 cells is considered troublesome .
• a stable packaging cell which expresses adenovirus region El, and E2a, for example derived from adenovirus tsl25.
• E2a for example derived from adenovirus tsl25.
• functional expression of E2a can be timed to optimise the yield of recombinant AAV.
• the additionally required helper functions are provided in the form of an El, E2a deleted adenovirus or in the form of plasmid DNA containing the helper virus function encoding genes, whereas the helper adenovirus vector or the plasmid DNA contains no sequence overlap with the helper virus functions already present in the packaging cells of the present invention, leading the formation of RCA.
• extra E2a helper function may be supplied to the packaging cell provided that the method does not introduce sequence overlap with the El-region already present in the packaging cells, leading to the formation of RCA.
• the expression of adenovirus late genes is essentially repressed either by intervention with the transcription of the late genes or by removing one or more of the encoding genes from the DNA encoding the additionally required helper function.
• the cells of the invention are grown to large numbers for the production, harvesting and purification of recombinant AAV.
• the cells are supplied with the recombinant AAV DNA, the DNA containing the AAV rep- and cap- genes and DNA containing the helper virus functions.
• the AAV rep- and cap- genes are physically linked to the plasmid DNA providing the additionally required helper function such that they are present on one and the same molecule.
• the cells can be supplied with the DNA needed for the production of recombinant AAV just prior to the start of recombinant AAV production, in which case, for each production the cells need to be supplied with the DNA through a process.
• Said process can be any method suitable for the transfection or infection of DNA into large numbers of cells.
• the DNA required for the production of recombinant AAV is transfected into PER cells by means of poly (2- (dimethylamino) ethyl-10-4- aminobutyl) phosphazene or other poly (organo) phosphazenes .
• parts of the DNA required for the production of recombinant AAV can be stably integrated into the PER cell chromosomal DNA.
• the cells of the invention recombinant AAV is produced with the packaging cell of the invention growing in suspension cultures using completely defined serum free medium.
• a method for generating a packaging cell containing all necessary helper function for an AAV-reproductive cycle, whereby said helper functions do not contain overlap leading to the formation of replication competent helper virus is provided.
• said packaging cell is stably transformed with the adenovirus El-region, which region does not contain overlap with additionally required helper functions.
• said packaging cell is stably transformed with the El-region and the E2a-gene. In this particular embodiment of the invention the E2a-function can be switched on or off at will following a signal.
• the E2a-gene is derived from adenovirus mutant H5tsl25 whereby said signal is a switch in temperature.
• said packaging cell is stably transformed with the adenovirus 5 El-region, the E2a gene and the adenovirus 5 VA-region (Martinez et al, 1989) or the adenovirus 5 E4orf6 gene, or both.
• the transcriptional activity of the adenovirus 5 VA-region and/or the adenovirus 5 E4orf6 gene is regulated. Meaning that the transcriptional activity can be switched on or off at will following a signal.
• additional required helper function also refers to helper virus functions allowing efficient (large scale) production of recombinant AAV for which the encoding genes are not stably integrated in the DNA of the recombinant AAV producing cell or for which additional expression is desired.
• additional required helper functions may be provided through any viral or non-viral method able to transfer foreign genetic material into mammalian cells such as but not limited to : poly (organo) phosphazenes, polyethylenimine, calcium phosphate precipitation, electroporation, recombinant, lipid or liposome mediated gene transfer.
• a packaging cell requiring only additional AAV-packaging function and a recombinant AAV-vector for the production of recombinant AAV.
• Said packaging cell comprises and provides the required adenovirus helper function from stably integrated adenoviral DNA.
• said helper function is provided by a stably integrated El- region.
• said helper function is provided by a stably integrated El-region and a stably integrated E2a gene.
• the E2a-function can be switched on or of at will following a signal.
• the E2a-gene is derived from adenovirus mutant H5tsl25 whereby said signal is a switch in temperature.
• said packaging cell is stably transformed with the adenovirus 5 El-region, the E2a gene and the adenovirus 5 VA-region (Martinez et al, 1989) or the adenovirus 5 E4orf6 gene, or both.
• the transcriptional activity of the adenovirus 5 VA-region and/or the adenovirus 5 E4orf6 gene is regulated, meaning that the transcriptional activity can be switched on or off at will following a signal.
• the invention provides a cell-culture comprising a cell according to the invention.
• a suspension culture or other large scale culture such as a bioreactor culture
• medium devoid of any human or animal constituents i.e. in serum free medium.
• Several systems have been devised to grow mammalian cells to large numbers. These include but are not limited to roller bottle culture, cell cubes and bioreactors. Each of these systems has advantages and disadvantages. Bioreactors in which cells are grown in suspension are the easiest to standardise and to scale to increasingly larger volumes. However, one drawback is that cells in suspension are not easily transfected. Many different cell culture media are developed to support optimal growth of a large variety of different cells.
• DMEM Dulbecco' s modified Eagles medium
• bovine serum bovine serum
• AAV-2 AAV serotypes
• other AAV serotypes such as 1 and 3 to 5
• serotypes can be adapted for the same purposes.
• dependoviruses common in other species can be used for the same purposes, for instance canine adeno- associated virus is able to infect human cells.
• human AAV replicates in many mammalian cell types as long as the species specific adenovirus is present, and dependoviruses from other species can be produced with the cells and methods of the present invention using the respective species specific adenovirus.
• Non-limiting examples of non-primate dependoviruses are avian- canine-, bovine adeno-associated virus (Berns, 1996).
• adenovirus 1 to 4, 6 to 51 or other human or animal adenoviruses can be manipulated for the same purpose provided that the function of the gene products is comparable.
• Gene products providing similar AAV helper function but which are derived from different viruses, such as but not limited to HSV, CMV and pseudorabies virus, or are derived from other natural sources or are produced in a synthetic form, can be used for the same purpose .
• Figure 1 Depicts the structure and the genome organisation of wt AV.
• Figure 2. PER.C6 cells were seeded at a density of 1 x 10 6 cells per 25 cm 2 tissue culture flask and cultured at either 32-, 37- or 39°C. At the indicated time points, cells were counted in a Burker cell counter. PER.C6 grows well at both 32-, 37- and 39°C.
• FIG. 3 Western blot with 35mg whole cell extract from cell lines generated from PER.C6 transfected with either pcDNA3 (upper panel, lane 1), pcDNA3wtE2A (upper panel, lane 2), pcDNA3tsE2A (upper panel lanes 4-14; middle panel, lanes 1-13 and lower panel lanes 1-12) or PER.C6 cells transiently transfected with pcDNA3tsE2A (upper panel, lane 3) .
• the blot was probed with an antibody specific for the E2A gene product (B6 aDBP) and visualised using the ECL detection system. All PER.C6tsE2A cell lines express the tsE2A encoded temperature sensitive DBP protein.
• FIG. 4 The tsE2A expressing cell line PER. C6tsE2A. c5-9 was cultured in suspension in serum free Ex-cellTM. At the indicated time points, cells were counted in a Burker cell counter. The results of 8 independent cultures are indicated. PER.C6tsE2A grows well in suspension in serum free Ex- cellTMmedium.
• the packaging plasmid pIM45 (7.3 Kb) contains the AAV-2 rep and cap- genes (McCarty et al, 1991) and was a kind gift from Dr. S. Zolotukhin.
• pACV- ⁇ gal (8.3 kb) is a plasmid containing a CMV-LacZ expression cassette between AAV-ITRs and was a kind gift from Dr. J.A. Kleinschmidt .
• Plasmid pIG.ElA.ElB contains the Ad5 Ela and Elb genes (nucleotides 459 to 3510 of Ad5) under transcriptional control of the human PGK promoter and is described in WO/97/00326.
• Plasmid pE2a is another name for plasmid pcDNA3wtE2A described below
• Plasmid pE4orf6 was generated by inserting a 929 bp fragment encoding the Ad5 E4orf6 protein into the BamHI site of pCMV/neo (Hinds et al, 1990) .
• wild-type human adenovirus type 5 (Ad5) DNA was treated with Klenow enzyme in the presence of excess dNTPs . After inactivation of the Klenow enzyme and purification by phenol/chloroform extraction followed by ethanol precipitation, the DNA was digested with BamHI. This DNA preparation was used without further purification in a ligation reaction with pBR322 derived vector DNA prepared as follows: pBR322 DNA was digested with EcoRV and BamHI, dephosphorylated by treatment with TSAP enzyme (Life
• pBr/Ad.Cla-Bam (ECACC deposit P97082117) wt Adeno type 5 DNA was digested with Clal and BamHI, and the 20.6 kb fragment was isolated from gel by electro-elution .
• pBR322 was digested with the same enzymes and purified from agarose gel by Geneclean. Both fragments were ligated and transformed into competent DH5 ⁇ . The resulting clone pBr/Ad. Cla-Bam was analysed by restriction enzyme digestion and shown to contain an insert with adenovirus sequences from bp 919 to 21566.
• Cla-Bam was linearised with EcoRI (in pBR322) and partially digested with Aflll. After heat inactivation of Aflll for 20' at 65 °C the fragment ends were filled in with Klenow enzyme. The DNA was then ligated to a blunt double stranded oligo linker containing a Pad site (5'- AATTGTCTTAATTAACCGCTTAA-3' ) . This linker was made by annealing the following two oligonucleotides : 5'- AATTGTCTTAATTAACCGC-3' and 5' -AATTGCGGTTAATTAAGAC-3 ' , followed by blunting with Klenow enzyme.
• pBr/Ad.Bam-rITRpac#2 (ECACC deposit P97082120) and pBr/Ad.Bam-rITR#8 (ECACC deposit P97082121)
• Bam-rlTR about 190 nucleotides were removed between the Clal site in the pBR322 backbone and the start of the ITR sequences. This was done as follows: pBr/Ad.Bam-rlTR was digested with Clal and treated with nuclease Bal31 for varying lengths of time (2', 5', 10' and 15').
• nucleotide removal was followed by separate reactions on pBR322 DNA (also digested at the Clal site) , using identical buffers and conditions. Bal31 enzyme was inactivated by incubation at 75 °C for 10' , the DNA was precipitated and resuspended in a smaller volume TE buffer. To ensure blunt ends, DNAs were further treated with T4 DNA polymerase in the presence of excess dNTPs . After digestion of the (control) pBR322 DNA with Sail, satisfactory degradation (-150 bp) was observed in the samples treated for 10' or 15' . The 10' or
• pWE/Ad.Aflll-rlTR (ECACC deposit P97082116) Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts.
• a linker containing a unique Pad site was inserted in the EcoRI sites of pWE15 creating pWE15.Pac.
• the double stranded Pad oligo as described for pBr/Ad. Aflll-Bam was used but now with its EcoRI protruding ends.
• fragments were then isolated by electro- elution from agarose gel: pWE15.Pac digested with Pad, pBr/Ad.Aflll-Bam digested with Pad and BamHI and pBr/Ad.Bam- rITR#2 digested with BamHI and Pad. These fragments were ligated together and packaged using ⁇ phage packaging extracts (Stratagene) according to the manufacturer's protocol. After infection into host bacteria, colonies were grown on plates and analysed for presence of the complete insert. pWE/Ad. Af111-rITR contains all adenovirus type 5 sequences from bp 3534 (Aflll site) up to and including the right ITR (missing the most 3' G residue) .
• the construct pWE/Ad. ⁇ 5' is an example of a replicating molecule according to the invention that contains two adenoviral ITRs and all adenoviral sequences between bp 3510 and 35938, i.e., the complete adenoviral genome except for the El region and the packaging signal. . pWE/Ad. ⁇ 5' has been made in a cosmid vector background from three fragments. First, the 5' ITR from Ad5 was amplified using the following primers :
• ITR-EPH 5 ' -CGG-AAT-TCT-TAA-TTA-AGT-TAA-CAT-CAT-CAA-TAA-TAT- ACC-3'
• ITR-pIX 5' -ACG-GCG-CGC-CTT-AAG-CCA-CGC-CCA-CAC-ATT-TCA-GTA- CGT-ACT-AGT-CTA-CGT-CAC-CCG-CCC-CGT-TCC-3' .
• the resulting PCR fragment was digested with EcoRI and Ascl and cloned into vector pNEB193 (New England Biolabs) digested with the same enzymes.
• the resulting construct was named pNEB/ITR-pIX. Sequencing confirmed correct amplification of the Ad5 sequences in the left ITR (Ad5 sequences 1 to 103) linked to the pIX promoter (Ad5 sequences 3511 to 3538) except for a single mismatch with the expected sequence according to GenBank (Accession no.: M73260/M29978 ) , i.e., an extra G- residue was found just upstream of the Aflll site.
• This ITR- pIX fragment was then isolated with EcoRI and Aflll and ligated to a EcoRI-Aflll vector fragment containing Ad5 sequences 3539-21567. The latter fragment was obtained by digestion of pBr/Ad.
• This cosmid is essentially the same as pWE/Ad.
• Af111-rITR ECACC deposit P97082116 apart from a deletion of the coding region of E2A.
• Deletion of the E2A coding sequences from pWE/Ad.Aflll-rlTR has been accomplished as follows.
• the adenoviral sequences flanking the E2A coding region at the left and the right hand side were amplified from the plasmid pBR/Ad. Sal . rITR (ECACC deposit P97082119) in a PCR reaction with the Expand PCR system (Boehringer) according to the manuf cturers protocol.
• the following primers were used:
• pVA (3.7 kb) is a pUC119 plasmid containing the VAI and VAII region of adenovirus 5 (nucl. 10555 until 11075) .
• the VA- genes of adenovirus 5 were cloned following PCR on isolated DNA from wild type adenovirus 5 using the primers 5'- ACGCGTCGACCTCTGGCCGGTCAGGCGCGCGCAA-3' and 5'- ACGCGGATCCCGCATCTGCCGCAGCACCGGATGC-3' .
• the PCR was performed using expand long templateTM PCR kit (Boehringer) according to the specifications of the manufacturer.
• the resulting fragment was digested with Sail and BamHI, present in the primers, and ligated into Sail, BamHI digested pUC119.
• PER.C6 cells were cultured in Dulbecco' s modified Eagle's medium (DMEM, Life technologies Breda, The Netherlands) containing 10% heat inactivated foetal bovine serum at 37 °C and 10% C0 2 .
• DMEM Dulbecco' s modified Eagle's medium
• Adherent cultures of PER.C6 cells were grown in DMEM supplemented with 10% foetal bovine serum and MgCl 2 (10 mM) at 37 °C and 10% C0 2 .
• Transfection of monolayer cultures HeLa cells and 293 cells were transfected using the Calcium Phosphate transfection system (Life technologies, Almere) according the specifications of the manufacturer. Monolayers of PER.C6 cells were transfected using LipofectAMINETM (Life technologies, Breda) according the specifications of the manufacturer . Transfection of suspension cul tures : PER.C6 cells in logarithmic growth phase were collected by centrifugation (3000g, 5 minutes, rt) . The cells were resuspended in transfection mix (described below) at a concentration of 2 x 10 6 cells per ml and incubated for three hours at 37 °C, 10% C0 2 .
• transfection were performed under continuous shaking (100 RPM).
• DMRIE-CTM Life technologies, Breda
• the transfection mix was made in DMEM according to the specifications of the manufacturer. After a three hour incubation in transfection mix the cells were collected by centrifugation (3000g, 5 min. rt) and resuspended in fresh Ex-CellTM medium to a final concentration of 10 6 cells per ml.
• Transfection with FuGENETM 6 (Boehringer Mannheim) was accomplished with transfection mix made in Ex-CellTM medium according to the specifications of the manufacturer. After a three hour incubation with transfection mix the cells were diluted with Ex-CellTM medium to a final concentration of 10 6 cells per ml.
• X l r X 2 are -N-CH 2 -CH 2 -N ( CH 3 ) 2 or -N-CH 2 - CH 2 -CH 2 -CH 2 -NH 2 .
• Transfection mixes were made by adding the indicated amount of PPZ to 500 ul Ex-CellTM medium. This solution was mixed with the same volume of Ex-CellTM containing the indicated amount of DNA. The mixture was incubated for one hour and was subsequently used to resuspend a pellet of 2 x 10 6 PER.C6 cells. The cells were incubated with the transfection mix for three hours and subsequently diluted with Ex-CellTM medium to a final concentration of 10 6 cells per ml. Transfected cells were harvested after 48 hours and analysed for ⁇ -galactosidase activity.
• Cells were stained for ⁇ -galactosidase activity with two different methods. For histoche ical analysis and determination of the number of infectious units the following procedure was used. Cells were washed twice with PBS (NPBI, Emmer-Compascuum) and fixed for 10 minutes in 0.2% glutaraldehyde (Sigma, Zwijndrecht, The Netherlands) in PBS.
• HeLa cells were seeded 4 x 10 4 cells per cm 2 .
• the medium was replaced the following day with fresh medium containing serial dilutions of rAAV and adenovirus tsl49 (20 pfu/cell). After 4 hours the medium was replaced by fresh medium and the cells were incubated for 24 hours at 37 °C, 10% C0 2 before the ⁇ -galactosidase staining.
• the titer of the recombinant AAV stock was calculated by counting the number of blue cells from the highest dilution giving rise to blue cells and multiplying this number by the dilution factor.
• adenovirus helper plasmid When more than one adenovirus helper plasmid was used, equal amounts (w/w) of the different adenovirus helper plasmids were used.
• Recombinant AAV production on adherent PER.C6tsE2A.c5-9 were performed as described for the PER.C6 cell line except for some modifications.
• the cell line was grown at 39 °C, 10% C0 2 . Before transfection the cells were seeded at 39 °C, 10% C0 2 such that they reached 70% confluency the next day. The cells were subsequently cultured for one day at 32 °C, 10% C0 2 . Next the cells were transfected at 37°C, 10% C0 2 as described for the PER.C6 cell line.
• Recombinant AAV was harvested 48 hours after transfection. The cells were scraped in their culture medium and subjected to three freeze thaw cycles. The cell debris was centrifuged (2000 RPM, 10 minutes, rt) . When using adenovirus tsl49 or El-deleted adenovirus vectors, the supernatants were heat inactivated at 56 °C for 1 hour. When adenovirus DNA fragments were used to complement AAV- production the supernatants were not heat inactivated. All supernatants were filtered (0.45 uM, Millipore) before storage at -20 °C .
• the producer cell lines complement for the El and E2A deletion from recombinant adenoviral vectors in trans by constitutive expression of the El and E2A genes, respectively.
• the pre- established Ad5-El transformed human embryo retinoblast cell line PER.C6 (WO 97/00326) and Ad5 transformed human embryo kidney cell line 293 (Graham et al, 1977b) were further equipped with E2A expression cassettes.
• the adenoviral E2A gene encodes a 72 kDa DNA Binding Protein (DBP) which has a high affinity for single stranded DNA. Because of this feature, constitutive expression of DBP is toxic for cells.
• the tsl25E2A mutant encodes a DBP which has a Pro—»Ser substitution of amino acid 413 (Vliet van der et al, 1975) . Due to this mutation, the tsl25E2A encoded DBP is fully active at the permissive temperature of 32°C, but does not bind to ssDNA at the non-permissive temperature of 39°C. This allows the generation of cell lines that constitutively express E2A which is not functional and is not toxic at the non-permissive temperature of 39°C, but becomes functional after a temperature switch to the permissive temperature of 32°C.
• pcDNA3wtE2A The complete wildtype early region 2A (E2A) coding region was amplified from the plasmid pBR/Ad.Bam-rlTR (ECACC deposit P97082122) with the primers DBPpcrl and DBPpcr2 using the ExpandTM Long Template PCR system according to the standard protocol of the supplier (Boehringer Mannheim) .
• PCR was performed on a Biometra Trio Thermoblock, amplification program: 94°C for 2 minutes, 1 cycle; 94°C for 10 seconds + 51°C for 30 seconds + 68°C for 2 minutes, 1 cycle; 94°C for 10 seconds + 58°C for 30 seconds + 68°C for 2 minutes, 10 cycles; 94°C for 10 seconds + 58°C for 30 seconds + 68°C for 2 minutes with 10 seconds extension per cycle, 20 cycles; 68°C for 5 minutes, 1 cycle.
• the primer DBPpcrl CGG GAT CCG CCA CCA TGG CCA GTC GGG AAG AGG AG (5' to 3' ) contains a unique BamHI restriction site (underlined) 5' of the Kozak sequence (italic) and start codon of the E2A coding sequence.
• the primer DBPpcr2 CGG AAT TCT TAA AAA TCA AAG GGG TTC TGC CGC (5' to 3') contains a unique EcoRI restriction site (underlined) 3' of the stop codon of the E2A coding sequence.
• the bold characters refer to sequences derived from the E2A coding region.
• pcDNA3tsE2A The complete tsl25E2A coding region was amplified from DNA isolated from the temperature sensitive adenovirus mutant H5tsl25 (Ensinger and Ginsberg, 1972; Vliet van der et al, 1975) .
• the PCR amplification procedure was identical to that for the amplification of wtE2A
• the PCR fragment was digested with BamHI /EcoRI and cloned into BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3tsE2A.
• the integrity of the coding sequence of wtE2A and tsE2A was confirmed by sequencing.
• DMEM Fetal Bovine Serum
• FBS Fetal Bovine Serum
• lOmM MgCl 2 in a 10% C0 2 atmosphere at either 32°C, 37°C or 39°C.
• a total of 1 x 10 6 PER.C6 cells were seeded per 25cm 2 tissue culture flask (Nunc) and the cells were cultured at either 32°C, 37°C or
• the cells were transfected with 3-, 5- or 8 ⁇ g of either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINE PLUSTM Reagent Kit according to the standard protocol of the supplier (GIBCO BRL) , except that the cells were transfected at 39°C in a 10% C0 2 atmosphere. After the transfection, the cells were constantly kept at 39°C, the non-permissive temperature for tsl25E2A.
• the E2A expression levels in the different cell lines were determined by Western blotting.
• the cell lines were seeded on 6 well tissue culture dishes and sub-confluent cultures were washed twice with PBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS, supplemented with ImM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor) . After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein concentrations were determined by the Bio-Rad protein assay, according to standard procedures of the supplier (BioRad) .
• Equal amounts of whole-cell extract were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the ⁇ DBP monoclonal antibody B6 (Reich et al, 1983) .
• the secondary antibody was a horseradish-peroxidase conjugated goat anti mouse antibody (BioRad) .
• the Western blotting procedure and antibody incubations were performed according to the protocol provided by Millipore. The antibody complexes were visualised with the
• FIG. 3 shows that all of the cell lines derived from the pcDNA3tsE2A transfection express the 72-kDa E2A protein (upper panel, lanes 4-14; middle panel, lanes 1-13; lower panel, lanes 1-12) . In contrast, the only cell line derived from the pcDNAwtE2A transfection did not express the E2A protein (lane 2) . No E2A protein was detected in extract from a cell line derived from the pcDNA3 transfection (lane 1), which serves as a negative control. Extract from PER.C6 cells transiently transfected with pcDNA3tsl25 (lane 3) served as a positive control for the Western blot procedure. These data confirm that constitutive expression of wtE2A is toxic for cells and that this toxicity can be circumvented by using the tsl25 mutant of E2A.
• Cells were transfected with 7.2 ⁇ g of either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA using the Calcium Phosphate Transfection System according to the standard protocol of the supplier (GIBCO BRL) . Two days post transfection, cells were put on selection medium, i.e. DMEM supplemented with 10% FBS, lOmM MgCl 2 and 0.1 mg/ml G418. The first colonies appeared at day 12 post transfection.
• selection medium i.e. DMEM supplemented with 10% FBS, lOmM MgCl 2 and 0.1 mg/ml G418.
• Ad5.dl 802 is an Ad 5 derived vector deleted for the major part of the E2A coding region and does not produce functional DBP (Rice and Klessig, 1985).
• Ad5.dl802 was used to test the E2A trans-complementing activity of PER.C6 cells constitutively expressing tsl25E2A.
• Parental PER.C6 cells or PER.C6tsE2A clone 3-9 were cultured in DMEM, supplemented with 10% FBS and lOmM MgCl 2 at 39°C and 10% C0 2 in 25 cm 2 flasks and either mock infected or infected with Ad5.dl802 at an m.o.i. of 5. Subsequently the infected cells were cultured at 32°C and cells were screened for the appearance of a cytopathic effect (CPE) as determined by changes in cell morphology and detachment of the cells from the flask.
• CPE cytopathic effect
• Table 3 shows that full CPE appeared in the Ad5.dl802 infected PER.C6tsE2A clone 3-9 within 2 days. No CPE appeared in the Ad5.dl802 infected PER.C6 cells or the mock infected cells. These data show that PER.C6 cells constitutively expressing tsl25E2A complement in trans for the E2A deletion in the Ad5.dl802 vector at the permissive temperature of 32 C.
• PER cells as producer cells for recombinant AAV.
• PER cells are derived from human retina cells. The retina is not known for its ability to sustain AAV replication. We therefore verified whether PER cells are permissive for recombinant AAV production.
• PER.C6 cells were transfected using LipofectAMINETM with the packaging plasmid pIM45, the rAAV-vector pACV- ⁇ gal (ratio 10:1 w/w) and infected with adenovirus tsl49. Recombinant AAV was isolated after two days and titrated on adenovirus infected HeLa cells.
• pACV- ⁇ gal produced on PER.C6 cells had a titer of 2 x 10 7 infectious units (IU) per ml or 20 IU per cell.
• the yield of virus per cell obtained with this system is comparable or better then those reported for 293 cell lines with the packaging plasmid pIM45 (Vincent et al, 1997) .
• PER.C6 cells were transfected with the packaging plasmid pIM45, the rAAV-vector pACV- ⁇ gal (ratio 10:1 w/w) and infected with an El-deleted adenovirus vector IG. Ad.MLP . Luc (Vincent et al, 1996) .
• the yield of rAAV using the El deleted adenovirus vector IG. Ad.MLP. Luc was 2 x 10 7 IU/ml and thus the same as with adenovirus tsl49. This result indicates that both the pattern and the level of El-expression in PER.C6 is allowing for the efficient production rAAV.
• the construct pWE/Ad.Af111-rITR does, as mentioned contain all adenovirus except El, however, the promoter of the protein IX gene contains a deletion.
• pWE/Ad.D5' contains the left ITR and the full length protein IX promoter.
• Recombinant AAV could be produced with both adenovirus DNA fragments. The yields of recombinant AAV were different with both constructs. The yield of recombinant AAV using pWE/Ad.Af111-rITR was significantly higher than using pWE/Ad.D5' (Table 4).
• One reason for the difference in yield could be differences in expression of relevant proteins from the two different plasmids. Another reason could be that expression of adenovirus 5 protein IX negatively affects recombinant AAV production.
• transfection reagents specifically the liposomes
• transfection of PER.C6 cells growing in suspension could, however, be achieved by using the non- liposomal reagent FuGENETM 6 or by avoiding the contact of the liposome:DNA complex (DMRIE-C) with the Ex-CellTM medium (Table 5) .
• Alternative transfection agents are poly (organo) phosphazenes . Not much is known about the ability to transfect cells with these agents.
• PER cells for the large scale production of recombinant AAV.
• Large scale production potential was first evaluated on adherent PER.C6 cells.
• Ten 170 cm 2 (Greiner) dishes were seeded with 2 x 10 7 PER.C6 per dish in DMEM + 10% FCS .
• Cells were transfected with pACV- ⁇ gal, pIM45 and pWE/Ad.Af111-rITR (2 : 8 : 30 ugram respectively) .
• the cell suspension was freeze thawed twice and subsequently incubated with DNasel (100 ugram/ml) at 37 °C 30 minutes. The suspension was subjected to two additional freeze thaw cycles after which the cell debri was removed by centrifugation (3000 RPM, 10 minutes) . The supernatant was incubated with 13.3 ml of saturated (NH 4 ) 2 S0 4 (4 °C, 10 minutes) . The precipitate was removed by centrifugation at 10.000 RPM in an SW27.1 rotor (4 °C, 15 minutes). Supernatant was incubated with an additional 26.6 ml of saturated (NH 4 ) 2 S0 4 and incubated for 20 minutes at 4 °C .
• the virus was pelleted by centrifugation at 12.000 RPM in an SW27.1 rotor (4 °C, 30 minutes) .
• the pellet was resuspended in 5 ml PBS (NPBI) and divided equally over two Quick-Seal Ultra-Clear tubes (Beckman Instruments, Mijdrecht, The Netherlands) .
• the virus suspension was underlayed with an equal volume of OptiPrep (Nycomed Pharma AS, Oslo, Norway) .
• the sealed tubes were rotated (20 minutes, 10 RPM) at an angle of 80 degrees.
• the virus was separated by density centrifugation (3 hours at 71.000 RPM) in an VTi80 rotor (Beckman Instruments).
• Experiment 1 was performed in 6 well dishes in stationary cultures.
• Experiment 2 was performed in 6 well dishes in shaking cultures (100 RPM) . During the transfection the cultures where incubated without shaking.
• Experiments 3 and 4 were performed in Erlenmeyer cultures under continuous shaking (100 RPM).
• Parvoviridae The viruses and their replication. In: Fields Virology, Lippincott - Raven, New York
• J Gen Virol 36 59-72 Graham FL, Smiley J, Russell WC, Naiva R (1977b) Characteristics of a human cell line transformed by DNA from adenovirus type 5. J Gen Virol 36: 59-72

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