WO2001020014A1 - Modified adenoviral vectors for use in gene therapy - Google Patents
Modified adenoviral vectors for use in gene therapy Download PDFInfo
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- WO2001020014A1 WO2001020014A1 PCT/NL2000/000640 NL0000640W WO0120014A1 WO 2001020014 A1 WO2001020014 A1 WO 2001020014A1 NL 0000640 W NL0000640 W NL 0000640W WO 0120014 A1 WO0120014 A1 WO 0120014A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Definitions
- the present invention relates to the field of human gene therapy, in particular to gene therapy vehicles with reduced expression of viral genes, more specifically with a reduced immunity.
- the invention provides novel expression vectors and complementing cell lines, including means and methods to produce such vectors and cell lines, and applications of such vectors, cell lines and methods in human gene therapy protocols.
- Non-viral gene systems for in vivo delivery into target cells include a variety of DNA-mediated methods, like direct injection of naked DNA and particle bombardment.
- DNA-mediated methods like direct injection of naked DNA and particle bombardment.
- alternative systems have been developed utilizing the entrapment of the DNA in vesicles (like liposomes) , or binding of the DNA to synthetic conjugates (like e.g. transferrin-polylysine) .
- conjugate-DNA complexes can be delivered via receptor- mediated endocytosis.
- Viral gene-transfer systems are based on the natural capability of viruses to deliver their genes in mammalian cells.
- the high gene-transfer efficiency of viruses has led to the development of viral vectors in which part of the viral genome has been replaced by a transgene to be introduced into eukaryotic cells.
- the most commonly used viral systems are based on retroviruses , adenoviruses (Ad) and adeno-associated viruses.
- Ad adenoviruses
- Ad adeno-associated viruses.
- Each of these viral delivery systems has its own characteristics in terms of efficiency of gene delivery, integration capability, maximum insert size of the recombinant (recombinant) gene, vector yields, stability of expression, etc. Such characteristics may determine the suitability of a certain delivery system for a specific gene therapy protocol (Bout, 1997) .
- Retroviruses and adeno- associated viruses have been a focus for development because of their capacity to stably integrate DNA sequences into chromosomes of the target cells.
- the advantage of adenoviruses is their ability to mediate efficient expression of the transgene in a variety of cells, including post- mitotic and/or non-dividing cells, as well as the ease with which these viruses can be propagated and purified to very high titers.
- Inherent to all gene transfer methods is the presentation of foreign antigenic material on the target cell, derived from the vehicle and/or from the transgene encoded product.
- Immune effectors have been identified as cytotoxic T lymphocytes (CTLs) , which destroy vector-transduced cells, as well as B cells— which secrete neutralizing antibodies that can block repeated gene transfer.
- CTLs cytotoxic T lymphocytes
- CTLs continuously monitor the cells and tissues of the body in search of cells synthesizing foreign or abnormal proteins.
- the recognition of virus- infected cells by CTLs requires fragments (i.e. peptides) of foreign antigens that are presented at the cell surface in association with class I molecules of the major histocompatibility complex (MHC; for a recent review see Ploegh et al . , 1998) .
- MHC major histocompatibility complex
- the majority of these peptides are generated in the cytosol of virus-infected cells by degradation of poly-ubiquitinated viral proteins.
- the resulting viral peptides are transported from the cytosol to the endoplasmic reticulum (ER) , through the action of the ATP dependent transporter for antigen presentation (TAP) complex.
- ER endoplasmic reticulum
- TAP antigen presentation
- MHC class I heavy chains assemble with ⁇ 2 microglobulin and peptide into stable hetero-trimeric MHC class I complexes that are transported via the secretory route to the plasma membrane.
- Their expression at the cell surface enables CTLs to play their decisive role in the antiviral defense.
- Ad vectors that are deleted for the El and E2A region.
- E2A E2A encoded DNA binding protein
- DBP DNA binding protein
- the tsDBP is functionally active at the permissive temperature (32°C) , whereas it is nonactive at the nonpermissive temperature (39°) .
- tsDBP is not toxic at the nonpermissive temperature.
- PER/E2A a new cell line, designated PER/E2A, that constitutively expresses high levels of the tsE2A gene. This cell line can easily be cultured at the nonpermissive temperature.
- functionally active DBP is needed, e.g., for replication of E2A-deleted Ad vectors, the cells can be incubated at the permissive temperature.
- the PER/E2A cell line and the E1/E2A deleted vectors were designed such that overlap between the Ad sequences in the cell line, i.e. El and E2A coding adenoviral sequences, and sequences in the recombinant Ad vector was excluded, at least to the extent that may lead to homologous recombination between vector DNA and adenoviral sequences present in the complementing cell line, which could lead to the formation of reverted viruses that have recaptured the El and/or E2A genes.
- E2A The deletion of the E2A gene eliminated the residual expression of E2A and the expression of Ad late genes, e.g., penton-base and fiber ( Figure 1A) .
- these vectors still expressed significant amounts of the E4 , e.g. E4-orf6, and E2B genes, e.g., pTP ( Figure IB) .
- Transcription of E4 may even be up regulated in the absence of DBP since the E4 promoter is a natural target for repression by DBP (Blanton and Carter, 1979; Nevins and Winkler, 1980; Chang and Shenk, 1990) .
- cells infected by E1/E2A deleted vectors still produce and present non-self antigens, when delivered to humans and may be eradicated from the recipient by the immune system.
- the present invention provides modifications to E2B and/or E4 regions or regions controlling the E2B and/or E4 regions in adenoviral vectors and/or in the complementing cell lines therefor.;
- E4 constitutes approximately 10% of the total length of the Ad genome.
- Several differentially spliced mRNAs are synthesized from the E4 region during infection and are predicted to code for seven different polypeptides, six of which have been identified in infected cells.
- E4 encoded proteins have an important function in virus growth in cultured cells since mutant viruses that lack the entire E4 region have a severe defect in replication (Weinberg and Ketner, 1986; Huang and Hearing, 1989) . Such E4 lacking viruses display defects in viral DNA replication, viral late mRNA accumulation, viral late protein synthesis, and the shut-off of host cell protein synthesis. Although the exact function of all individual E4- encoded polypeptides has not been defined to date, mutagenesis of individual open reading frames (orfs) has shown that multiple products encoded by E4 are functionally compensatory. Thus, it was found that either the E4-orf3 or the E4-orf6 product is prerequisite for virus replication in cultured cells whereas the other E4 products are dispensable (Huang and Hearing, 1989) .
- E4-orf3 as well as E4-orf6 encoded proteins are (independently) involved in post-transcriptional processes that increase viral late protein synthesis. They do so by facilitating the cytoplasmic accumulation of the mRNAs encoding these proteins (Sandier and Ketner, 1991) . Moreover, they maintain the nuclear stability of unprocessed pre-mRNAs transcribed from the major late promoter, presumably by affecting the splicing of late RNAs . This leads to an expansion of the pool of late RNAs available for maturation and transport to the cytoplasm.
- the E4-orf6 encoded 34 kDa protein forms a complex with the E1B 55 kDa protein that selectively increases the rate of export of viral late mRNAs from the nucleus.
- the E4orf6-34 kDa/ElB-55 kDa complex is located in so called viral inclusion bodies, the region where viral DNA replication, viral late gene transcription and RNA processing occur (Pombo et al . , 1994) .
- both the E4-orf6 and E4-orf3 encoded proteins are required for Ad DNA synthesis.
- E4 protein products are involved in controlling the cellular transcription factor E2F as well as the phosphorylation of cellular (and viral) proteins.
- E4-orfl and E4- orf6 products have oncogenic potential.
- the E4-orf6 protein either alone or in a complex with the ElB-55-kDa protein, binds the cellular protein p53 thereby blocking its potential to activate the transcription of tumor-suppressing genes (Dobner et al . , 1996; Moore et al . , 1996).
- the E4-orf6 protein may prevent the induction of apoptosis by p53.
- Ad vectors may further reduce viral genome replication and expression of early and late viral genes. This may, in turn, diminish the antigenicity of the vectors.
- vectors from which the E4 function is impaired can be considered safer than E4 -containing vectors because they do not express E4 -encoded proteins that can induce oncogenesis or which are toxic to the host cell.
- the impairment of the E4 function within the Ad vector backbone can influence the activity of the promoter that drives the expression of the transgene.
- CMV cytomegalovirus
- RSV Rous sarcoma virus
- mammalian promoters such as the elongation factor-1 alpha (EF-l ⁇ ) or the ubiquitin-C (UbC) promoter, can mediate persistent expression of the transgene from an Ad vector that does not produce E .
- EF-l ⁇ elongation factor-1 alpha
- UbC ubiquitin-C
- E2 The early region 2 (E2) of Ad encodes three gene products that are required for Ad DNA replication.
- E2 can be divided into two transcription units, E2A and E2B. Both E2A and E2B are transcribed from the same promoter region, designated the E2 promoter.
- E2 is transcribed from the E2 -early promoter located at 76 map units.
- the transcription switches to another promoter, the E2-late promoter located at 72 map units (reviewed by Swaminathan and Thimmapaya, 1995) .
- Transcription initiation from the E2 -early promoter is strongly induced by the polypeptides encoded by El, which is mediated via E2F and jun/ATF transcription factors. Transcription initiation from the E2-late promoter is less well understood. This promoter consists of a TATA- like sequence, SP-1 binding sites and a CAAT box.
- the E2-late promoter is not regulated by proteins of the E1A region.
- the E2A region encodes the 72kDa single stranded DNA binding protein (DBP) . It plays a pivotal role in both the initiation and elongation of Ad DNA replication (reviewed by van der Vliet, 1995 and references therein) .
- DBP is thought to increase the affinity of the host cell nuclear factor 1 (NF1) to the auxiliary region of the inverted terminal repeat (ITR) of the Ad genome. This, in turn, facilitates binding of the pTP/pol complex (see below) to the core region of the ITR.
- DBP stimulates the NF1 dependent formation of pTP-dCMP, which forms the DNA- replication initiation complex.
- DBP facilitates Ad DNA template unwinding by destabilization of the DNA helix in the replication fork. Following unwinding, DBP binds cooperatively to the single stranded Ad DNA in a non-sequence specific manner, thereby forming a protein chain at the displaced strand. This may be the mechanism by which DBP destabilizes the duplex DNA ahead of the replication fork. Fourthly, DBP prevents intramolecular renaturation of the ITRs of the displaced DNA strand, whereas it facilitates intermolecular renaturation of two displaced strands of opposite polarity, originating from initiation of DNA replication at different molecular ends. Finally, DBP has a positive regulatory effect on the activity of the major late promoter, the promoter that drives expression of the Ad late genes (Chang and Shenk, 1990) .
- E2B-specific mRNAs that are derived from differential splicing. They encode two polypeptides, the 80 kDa pTP and the 140 kDa DNA polymerase (pol) that form a stable heterodimer.
- the pTP/pol complex is recruited to the ITR by NF1, where it binds to the core region and forms the pre-initiation complex.
- the ITR partially unwinds and the replication-initiation reaction is primed, i.e. a pTP-dCMP coupling takes place that is catalyzed by pol. This is followed by synthesis of a pTP-CAT intermediate at G4-T5-A6 of the ITR.
- the pTP-trinucleotide intermediate jumps back to the G1-T2-A3 position of the ITR.
- Elongation of the Ad DNA synthesis starts after dissociation of DNA-bound pTP from pol.
- the elongation reaction is enhanced by DBP, as discussed above, and progeny DNA accumulates.
- the DNA-bound pTP as well as free pTP are proteolytically cleaved into TP by the Ad protease late in the infection cycle.
- the proteolytic maturation of free pTP destroys its capacity to function as a primer for DNA replication and thus the initiation of new DNA replication cycles is stopped.
- Mature TP that is covalently bound to newly synthesized Ad DNA protects the DNA from exonuclease activity and is involved in the attachment of the viral DNA to the nuclear matrix. Finally, DNA-bound TP stabilizes the incoming pTP/pol heterodimer at the core region of the ITR during the initiation of DNA replication in the next lytic infection cycle.
- E4orf6 34 kDa and E4orf3 11 kDa are considered to be highly toxic when constitutively synthesized in a complementing cell, meaning that the expression of the corresponding genes needs to be regulated tightly. This generally leads to complementing cells that do not support manufacturing of high titer batches of the respective recombinant Ad vectors (Lusky et al . , 1998 and references therein) .
- deletion approach is not suitable for the production of a homogeneous population of E2B- crippled Ad vectors.
- recombinant Ad vectors that are deleted of some or even all viral coding sequences can only be propagated using a helper Ad that supports replication and packaging of such vectors by providing all the necessary proteins in trans (Fisher et al . , 1996; Hardy et al . , 1997; Kochanek et al . , 1996; Kumar-Singh and Chamberlain, 1996).
- helper Ad that supports replication and packaging of such vectors by providing all the necessary proteins in trans
- the packaging sequence of such a helper virus is flanked by lox sites which are targets for the CRE- recombinase.
- the packaging signal is excised from the helper vector when the packaging cells (used for production and packaging of the gutted vectors) express the CRE- recombinase, thereby preventing the packaging of the helper vector.
- This system has serious limitations with respect to the efficiency of excision of the packaging signal and to the low yields of the gutted vector. Therefore, the crude vector batches produced in this way are contaminated with helper virus that is formed due to inefficient excision of the packaging signal. Moreover, the removal of this contaminating helper virus is laborious and incomplete, which means that it is practically impossible to obtain a helper virus free vector batch.
- a final problem is that extensive deletion of the coding sequences from the Ad genome renders the virus unstable and leads to rearrangement of the viral DNA during replication (Parks and Graham, 1997) . This is presumably due to fact that genomes smaller than 75% of the wild-type genome are inefficiently packaged into virus particles. To circumvent this problem, the deletion of large parts of the viral genome has to be compensated by addition of heterologous sequences to increase the net size of the vector genome. Such an approach has the intrinsic risk of introduction of unintentional, and perhaps yet unknown cryptic transcriptional signals and open reading frames within the vector backbone and increases the risk of (homologous) recombination with the cellular DNA during replication.
- the present invention now provides a method for producing a recombinant adenovirus-like gene delivery vehicle having reduced expression of adenoviral E2B and/or E4 gene products in a target cell for gene therapy, comprising generating a recombinant adenoviral vector lacking E1A and preferably E1B sequences, but having at least the E2B and/or E4 sequences encoding products essential for adenoviral replication, wherein said E2B and/or E4 sequences have been modified to lead to a reduced expression and/or induced expression of at least one of said essential products.
- Modification in this respect means any change at the nucleic acid level that diminishes the expression and/or the function of any of the gene products of the relevant genes, be it by mutation in a coding sequence or mutation in a regulatory sequence, whereby the expression is not completely or permanently deleted. It also means replacing a regulatory sequence of any of these genes by one or more inducible regulatory sequences, be it repressors, transactivation sites, inducible promoters or any other inducible sequence, whereby non-leaky ones are preferred. Combinations may be made to avoid leakage in the non-activated or repressed state. In a preferred embodiment at least open reading frame 1, 3 or 6 of E4 is so modified.
- E2B expression is at least in part reduced in said gene delivery vehicle.
- expression of E2B can be reduced directly by modifying E2B sequences or indirect by modifying E4 sequences or by both type of modifications.
- expression of E4 gene products can be reduced by modification of E4 sequences or by modification of E2B sequences or both.
- open reading frame 1, 3 or 6 of E4 are sequences that are essential for replication and/or sequences that lead to toxicity for target cells and/or complementing cells. Therefor down regulation or induction of such sequences is highly desired. Attenuation is preferably achieved through at least one mutation in at least an E2B and/or E4 promoter.
- the invention also provides a method as disclosed herein before, wherein said vector further comprises an E2B and/or an E4 promoter, wherein said E2B and/or E4 promoter are attenuated through a mutation therein.
- E2B and/or E4 is placed under control of at least one, preferably synthetic inducible promotor and/or repressor.
- Suitable inducible promoters are well known in the art. A couple of suitable and preferred ones are disclosed herein in the detailed description. Highly preferred inducible promoters are the ones that are described as synthetic, comprising e.g. an artificial TATA-box and a sequence capable of being recognized by a prokaryotic or similar transactivation signal. Typically a complementation cell would then be able to provide said signal through e.g. an expression cassette introduced therein.
- the vector also lacks a functional E2A region, which can be elegantly provided by a complementing cell, especially in the form of a temperature sensitive variant of E2A.
- a function of the E4 34 kDa protein can also be attenuated by inhibiting the binding to E1B 55kD.
- the invention also provides a method according wherein said vector lacks a sequence encoding E1B 55kD protein capable of binding an E4 34 kDa gene product.
- the vectors according to the invention are propagated in complemanting cells.
- the invention also provides a method as disclosed above, further comprising transducing a complementing cell with said recombinant adenoviral vector wherein said complementing cell provides all functions and/or elements essential for replication of said recombinant adenoviral vector, which are lacking in the genome of said vector.
- a gene delivery vehicle is defined as any viral particle derived from an adenovirus, a chimaeric adenovirus or comprising adenoviral elements, capable of infecting cells and delivering a gene there to.
- a chimaeric adenovirus may be a chimaera of two or more different adenoviruses, manipulated to give good infection and yet low antigenicity, etc. It may also be a chimaera of adenovirus with another virus such as AAV or a retrovirus, in order to be able to integrate a nucleic acid of interest into a host cell genome.
- the invention also provides a method wherein said complementing cell further comprises all necessary functions and/or elements essential for producing a recombinant adenovirus-like gene delivery vehicle comprising said recombinant adenoviral vector.
- the function of the complementing cell goes beyond replication of the vector and typically includes packaging of the vectors according to the invention, which should then typically possess a functional packaging signal .
- the complementing cell according to the invention preferably also comprises the capability to provide the induction of the E4 and/or E2B products.
- the invention provides a method wherein said cell further comprises an expression cassette encoding a proteinaceous substance capable of transactivating the inducible (synthetic) promoter on the vector, preferably a method wherein said proteinaceous substance comprises a DNA binding domain from a prokaryote or a lower eukaryote and/or a transactivator domain. More details are given in the detailed description.
- the invention thus provides a recombinant adenoviral vector lacking E1A and preferably E1B sequences, but having at least the E2B and/or E4 sequences encoding products essential for adenoviral replication, wherein said E2B and/or E4 sequences have been modified to lead to a reduced expression and/or induced expression of at least one of said essential products, said vector being obtainable as an intermediate in a method as disclosed above.
- the adenovirus-like gene delivery vehicles having reduced expression of adenoviral E2B and/or E4 gene products in a target cell for gene therapy, obtainable by a method as described herein.
- such a vector and/or vehicle preferably comprises a therapeutic sequence such as a gene encoding a therapeutic protein, an anti-sense sequence, etc.
- a therapeutic sequence such as a gene encoding a therapeutic protein, an anti-sense sequence, etc.
- Such a vector can subsequently be used to introduce the therapeutic protein, anti -sense sequence etc. in cells of a patient, for example (but not limited to) to correct a certain inherited or acquired disorder or for vaccination purposes.
- a recombinant adenoviral vector of the invention in gene therapy settings is that expression of a nucleic acid interest delivered to a host through said vector is prolonged compared to a adenoviral vectors of the art . This is not only due to improved capabilities to avoid the host immune system (in or outside a cell) but also to other factors . Another reason for the observed prolonged expression is that promoters that are commonly used for the expression of gene of interest in a gene therapy setting are at least in part protected from shut down of expression in the host. A non-limiting example of such a commonly used promoter is the CMV promoter.
- Hi 3 — .
- Hi H- 3 ⁇ Hi 0 H- - rt ⁇ rt ⁇ Hi Dd Dd H ⁇ 3 ⁇ h- 1 CL CD rt ⁇ 0 0 • 0 »
- H- rt TJ o CD > cr o O rt ⁇ ⁇ TJ CD TJ rt ⁇ LQ CL h- 1 3 rt ⁇ H- 0 rt • ⁇
- CD cr TJ 3 0 3 CD ⁇ ⁇ ⁇ H3 rt 0 Dd ⁇ Hi CL ⁇ - ⁇ - 3 3 0 ⁇ - rt H 1 CD CD 3
- the vectors according to the invention can also be applied for purposes other than gene therapy such as, but not limited to, functional characterization of gene products in vi tro and in vivo .
- the vectors according to the invention can be used for overexpression of a variety of known and novel genes in cell lines, tissues or animals in order to find genes that encode for proteins with a desired function such as, but not limited to, those that interfere with cell proliferation and differentation.
- a desired function such as, but not limited to, those that interfere with cell proliferation and differentation.
- the vectors according to the invention are replication defective and do express the remaining viral genes, if at all, only at background levels; interference of the vector with the function and the effect of the gene of interest is therefore at least in part prevented.
- the vectors according to the invention can also be used as a vaccine.
- the vectors according to the invention can be equipped with an expression cassette that codes for a protein against which an immune response has to be raised.
- the vectors according to the invention are replication defective and express the remaining viral genes, if at all, only at background levels, it is expected that the immune response will be directed primarily against the transgene product.
- the vectors according to the invention can also be used for protein production in mammalian cells. Therefore, the vectors according to the invention can be equipped with an expression cassette that encodes a protein of interest to be synthesized and processed in said cells. For this application, it is of importance that the vector itself does not interfere with the cellular metabolism which is harmful to the cell and which impairs synthesis and processing of the protein of interest . Because the vectors according to the invention are replication defective and express the remaining viral genes, if at all, only at background levels, said vectors are less toxic to the cells which, in turn, results in a prolonged synthesis of the protein of interest .
- Adenoviral vectors typically do not or very inefficiently integrate into the host cell genome. To increase at least in part the integration frequency of adenovirus vectors elements from integrating viruses can be included in the vector.
- the vectors and the gene delivery vehicles of the invention are suited for integrating vectors since the vectors are at least in part not toxic to the cells.
- the invention therefor provides a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to the invention, wherein said adenoviral vector comprises at least one adeno-associated virus terminal repeat or a functional equivalent thereof.
- said adenovirus vectors comprise at least two adeno-associated virus terminal repeat or a functional equivalent thereof.
- a functional equivalent of an adeno- associated virus terminal repeat comprises the same integrating function in kind not necessarily in amount.
- the adeno-associated virus terminal repeat is present at the extreme ends of the adenoviral vector.
- the invention further provides a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to the invention comprising elements derived from at least two different adenovirus serotypes .
- the invention further provides a method for ex vivo production of a gene product in a cell comprising providing CD 3 rt H ⁇ ⁇ ⁇ - CD Hi O TJ TJ rt Hj O
- Plasmid based system for the generation of recombinant Ad vectors that are deleted for El and/or E2A and in which the E4 and/or E2B promoter is replaced by a synthetic promoter
- 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 Ec ⁇ KV and BamHI , de- phosphorylated by treatment with TSAP enzyme (Life Technologies) and purified on LMP agarose gel (SeaPlaque GTG). After transformation into competent E. coli DH5a (Life Technologies).
- Sal -rlTR (ECACC deposi t P97082119)
- pBr/Ad.Bam-rlTR was digested with BamHI and Sail.
- the vector fragment including the adenovirus insert was isolated from LMP agarose (SeaPlaque GTG) and ligated to a 4.8 kb Sall-
- Sal-rlTR contains adeno type 5 sequences from the Sail site at bp 16746 up to and including the rITR (missing the most 3' G residue) .
- Clone pBr/Ad.Cla-Bam was linearized 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. After precipitation of the ligated DNA to change buffer, the ligations were digested with an excess of Pad enzyme to remove concatamers of the oligo.
- One clone that was found to contain the Pad site and that had retained the large adeno fragment was selected and sequenced at the 5' end to verify correct insertion of the Pad linker in the (lost) Aflll site.
- 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 minutes, the DNA was precipitated and re-suspended in a smaller volume TE buffer. To ensure blunt ends, DNA's were further treated with T4 DNA polymerase in the presence of excess dNTPs. After digestion of the (control) pBr322 DNA with Sail, satisfactory degradation
- 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.AfIII -BamHI was used but now with its EcoRI protruding ends.
- the following fragments were then isolated by electro- elution from agarose gel: pWE15.pac digested with Pad , pBr/AfIll-Bam digested with Pad and BamHI and pBr/Ad.Bam- rITR#2 digested with BamHI and Pad .
- 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) .
- G. Generation of pWE/Ad A fill -EcoRI pWE15.pac was digested with Clal and 5' protruding ends were filled using Klenow enzyme. The DNA was then digested with Pad and isolated from agarose gel.
- pWE/Af111-rITR was digested with EcoRI and after treatment with Klenow enzyme digested with Pad .
- the large 24-kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the C al-digested and blunted pWE15.pac vector using the Ligation Express tm kit from Clontech. After transformation of Ultra-competent XLIO-Gold cells
- pWE/Aflll-EcoRI contains Ad5 sequences from bp 3534-27336.
- the shuttle vector pAAO-E-TATA was constructed using the following primers:
- TATAplus 5 ' -AGC TTT CTT ATA AAT TTT CAG TGT TAG ACT AGT AAA TTG CTT AAG-3' TATAmin: 5 ' -AGC TCT TAA GCA ATT TAC TAG TCT AAC ACT GAA AAT TTA TAA GAA (The underlined sequences form a modified TATA box.)
- the primers TATAplus and TATAmin were annealed to yield a double stranded DNA fragment flanked by 5 ' overhangs that are compatible for ligation with Hindlll digested DNA.
- Hindlll digested pGL3-Enhancer Vector Promega
- tet3 5'-CCG GAG CTC CAT GGC CTA ACT CGA GTT TAC CAC TCC
- the amplified fragment was digested with Sstl and Hindlll (Sstl and Hindlll sites generated in the tet3 and tet5 primers are represented by the nucleotides in bold) and cloned into Sstl/Hindlll digested pAAO-E-TATA giving rise to pAAO-E-TATA-7xtetO. Sequence analysis confirmed the integrity of the heptamerized tet-operator sequence in the latter plasmid.
- pAAO-E-TATA-7xtetO was digested with Ncol , and the resulting fragment containing the 7xtetO sequence was purified from agarose and used in a ligation reaction with JVcoI digested pNEB.PmAs yielding pNEB . PmAs7xtetO.
- the plasmid pNEB.PmAs was obtained as follows: pBR/Ad.AflII-Bam (ECACC deposit P97082114) was digested with P el /Ascl and the resulting Pmel/ Ascl fragment containing the 5' end of the E2B region was purified from agarose and subcloned into Pmel /Ascl digested pNEB 193 (New England BioLabs Inc.) yielding pNEB.PmAs. Then, pNEB.
- PmAs7xtetO was digested with Ascl, Pmel and S al and the resulting Ascl/Pmel fragment (2808 bp) containing the 5' E2B region preceded by 7xtetO was purified from agarose using the Geneclean II kit, and used in a ligation reaction with Pmel/ Ascl digested pBR/Ad.AflII-Bam (the fragment that lacks the 5' end of the E2B region) yielding pBR/Ad.AflII-Bam. tetO-E2B .
- E2A coding sequences from pBR/Ad.Bam-rlTR (ECACC deposit P97082122) has been accomplished as follows.
- the adenoviral sequences flanking the E2A coding region at the left and the right site were amplified from the plasmid pBr/Ad.Sal-rlTR (ECACC deposit P97082119) in a PCR reaction with the Expand PCR system (Boehringer) according to the manufacturers protocol.
- the following primers were used:
- ⁇ E2A.BamHI 5 ' -GAG GTG GAT CCC ATG GAC GAG-3'
- the amplified D ⁇ A was digested with BamHI and Nsil (Nsil site is generated in the primer ⁇ E2A. DBP-stop, underlined) . Subsequently, the digested D ⁇ A fragments were ligated into SnaBl/Ba HI digested pBr/Ad.Bam-rlTR, yielding pBr/Ad.Bam- rITR ⁇ E2A.
- the unique Nsil site can be used to introduce an expression cassette for a gene to be transduced by the recombinant vector.
- pBR/Ad.Bam-rITR ⁇ E2A was digested with Ascl and Xbal and the resulting Asd/Xbal fragment containing the E2 promoter region was purified from agarose using the Geneclean II kit and subcloned into Ascl /Xbal digested p ⁇ EB 193 (New
- E2eSpeI 5' GGA CTA GTC TAA GTC TTC TCC AGC GGC CAC ACC CGG
- E2eSrf 5' GAG TTA TAC CCT GCC CGG GCG ACC GCA CC 3' E2lAat: 5' GGG CTG TGG ACG TCG GCT TAC CTT CGC AAG TTC GTA
- the amplification products of the E2 early and late promoter regions were purified from gel using the Geneclean II kit and digested with Spel. Thereafter, the Spel digested
- PCR products were ligated. After chloroform/phenol extraction, the ligated DNA's were digested with Aatll and
- the resulting DNA's were separated in an agarose gel and the ligation product that comprised the reconstituted and modified E2 (early and late) promoter was purified from agarose using the Geneclean II kit. The purified fragment was then used in a ligation reaction with Aatll/ Srfl digested pNEB-AX yielding pNEB-AX ⁇ E2p.
- the primers for amplification and mutation of the E2 promoter were chosen such that part of the native E2 early promoter spanning the E2F sites and a putative TATA-box was excluded from amplification.
- the modified E2 promoter region that is reconstituted by ligation of the two PCR products is deleted for the above mentioned promoter sequences.
- pNEB-AX ⁇ E2p was digested with Ascl and Srfl and the resulting Ascl/Srfl fragment containing the mutated E2 promoter was purified from agarose using the Geneclean Spin kit and cloned into Asd/Srfl digested pBR/Ad.Bam-rITR ⁇ E2A (the fragment that lacks the E2 promoter) giving rise to pBR/Ad.Bam-rITR ⁇ E2A ⁇ E2p.
- pBR/Ad.Bam-rlTR was digested with Pad and Sse83871 and the resulting Pacl/Sse83871 fragment containing the E4 region was purified from agarose using the Geneclean II kit and subcloned into Pacl/Sse83871 digested pNEB 193 (New
- pNEB-PaSe The latter plasmid was used to amplify the Ad sequences that flank the E4 promoter. Sequences upstream of the E4 promoter were amplified using the primers 3ITR and 5ITR whereas sequences downstream of the E4 promoter were amplified using the primers H3DE4 and AvDE4.
- 3ITR 5' CCG GAT CCT TAA TTA AGT TAA CAT CAT C 3'
- AvDE4 5 ' CTC CTG CCT AGG CAA AAT AGC 3 *
- nucleotides indicated in bold form unique restriction sites for Pad , Sstl , Hindlll and Avrll, respectively.
- PCR products were first purified using the QIAquick PCR purification kit.
- the PCR product of the sequence downstream of the E4 promoter was digested with Hindlll and Avrll.
- PCR product of the sequence upstream of the E4 promoter was digested with Sstl and ligated to a fragment that contained the 7xtetO sequence which was obtained by digestion of pAAO- E-TATA-7xtetO with Sstl and Hindlll.
- the ligation products were thereafter digested with Hindlll and Pad and the ligation product comprising the region upstream of the E4 promoter linked to the 7xtetO sequence was purified from gel.
- pNEB-PaSe.tetO was digested with Pad and Sse83871 and the fragment containing the artificial 7xtetO promoter sequences in front of the E4 region was purified from agarose and cloned into Pacl/Sse83871 digested pBR/Ad.Bam-rITR ⁇ E2A giving rise to pBR/Ad.Bam-rITR ⁇ E2A. tetO-E4.
- Notl /Pad fragment from pBR/Ad.Bam-rITR. ⁇ E2A. tetO-E4 was also cloned into Notl/Pad digested pBR/Ad.Bam-rITR. ⁇ E2A. ⁇ E2p (the fragment that lacks the E4 promoter region) giving rise to pBR/Ad. Bam-rlTR . ⁇ E2A. ⁇ E2p . tetO-E4.
- the cosmid pWE15.pac was linearized by digestion with Pad and used in a ligation reaction with BamHl/PacI digested pBR/Ad.Aflll-Bam. tetO-E2B and BamHI/ Pad digested pBR/Ad.Bam- rITR. ⁇ E2A. ⁇ E2p.
- the ligation mixture was used in a packaging reaction using ⁇ phage-packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII-rITR. ⁇ E2A.tetO-E2B.
- Pad-linearized pWE15.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam and BamHI/ Pad digested pBR/Ad.Bam-rITR-tetO-E4.
- the ligation mixture was used in a packaging reaction using ⁇ phage- packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII- rITR.tetO-E4.
- Pacl-linearized pWE15.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam and BamHI/PacI digested pBR/Ad.Bam-rITR. ⁇ E2A.
- the ligation mixture was used in a packaging reaction using ⁇ phage- packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII- rITR. ⁇ E2A.
- Pacl-linearized pWE15.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam and BamHI/PacI digested pBR/Ad.Bam-rITR. ⁇ E2A. tetO-E4.
- the ligation mixture was used in a packaging reaction using ⁇ phage-packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AfIII- rITR. ⁇ E2A.tetO-E4.
- Pacl-linearized pWE15.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AfIII-
- the ligation mixture was used in a packaging reaction using ⁇ phage-packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII-rITR. ⁇ E2A.tetO-E2B.tetO-E4.
- Adapter plasmid pMLP.TK (described in EP 95202213) was modified as follows: SV40 polyA sequences were amplified with primer SV40-1 (introduces a BamHI site) and SV40-2 (introduces a Bgrlll site) . In addition, Ad5 sequences present in this construct (from nt . 2496 to nt . 2779; Ad5 sequences nt . 3511 to 3794) were amplified with primers Ad5-1 (introduces a Bgrlll site) and Ad5-2.
- SV40 - 1 5 ' -GGGGGATCCGAACTTGTTTATTGCAGC-3 ' SV40 -2 : 5 ' -GGGAGATCTAGACATGATAAGATAC-3 ' Ad5-1 : 5 ' -GGGAGATCTGTACTGAAATGTGTGGGC-3 ' Ad5-2 : 5 ' -GGAGGCTGCAGTCTCCAACGGCGT-3 '
- PCR fragments were digested with Bglll and ligated.
- the ligation product was amplified with primers SV40-1 and Ad5-2 and digested with BamHI and Aflll.
- the digested fragment was then ligated into pMLP.TK predigested with the same enzymes.
- the resulting construct named pMLPI.TK, contains a deletion in adenovirus El sequences from nt . 459 to nt . 3510.
- This plasmid was used as the starting material to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged.
- a PCR fragment was generated from pZip ⁇ Mo+PyFlOl (N ⁇ ) template DNA (described in PCT/NL96/00195) with the following primers :
- LTR-1 5'-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA
- CAT AAC TG-3' and LTR-2 5 ' -GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC
- Pwo DNA polymerase (Boehringer Mannheim) was used according to manufacturers protocol with the following temperature cycles: once 5 minutes at 95°C; 3 minutes at 55°C; and 1 minute at 72°C, and 30 cycles of 1 minute at 95°C, 1 minute at 60°C, 1 minute at 72°C, followed by once 10 minutes at 72°
- the PCR product was then digested with BamHI and ligated into pMLPIO (Levrero et al . , 1991) digested with PvuII and BamHI, thereby generating vector pLTRlO.
- This vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter consisting of a part of the Mo-MuLV LTR having its wild-type enhancer sequences replaced by the enhancer from a mutant polyoma virus (PyFlOl) .
- the promoter fragment was designated L420. Sequencing confirmed correct amplification of the LTR fragment however the most 5 ' bases in the per fragment were missing so that the PvuII site was not restored.
- pLTRlO was digested with BstBI followed by Klenow treatment and digestion with Ncol .
- the HSA gene was obtained by PCR amplification on pUC18-HSA (Kay et al . , 1990) using the following primers:
- HSA1 5' -GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3 ' and
- pLTR-HSAlO was digested with EcoRI and BamHI after which the fragment containing the left ITR, packaging signal, L420 promoter and HSA gene was inserted into vector pMLPI.TK digested with the same enzymes and thereby replacing the promoter and gene sequences.
- AATTGTCTTAATTAACCGCTTAA-3 ' The ligation mixture was digested with Pad and religated after isolation of the linear DNA from agarose gel to remove concatamerised linkers. This resulted in adapter plasmid pAd5/L420-HSA.pac .
- pAd/L420-HSA was digested with Avrll and Bgrlll followed by treatment with Klenow to obtain blunt ends.
- the 5.1 kb fragment with pBr322 vector and adenoviral sequences was isolated and ligated to a blunt 1570 bp fragment from pcDNAl/amp (Invitrogen) obtained by digestion with Hhal and Avrll followed by treatment with T4
- This adapter plasmid was named pAd5/Clip.
- this plasmid was partially digested with EcoRI and the linear fragment was isolated. An oligo of the sequence 5' TTAAGTCGAC-3 ' was annealed to itself resulting in a linker with a Sail site and EcoRI overhang. The linker was ligated to the partially digested pAd5/Clip vector and clones were selected that had the linker inserted in the EcoRI site 23 bp upstream of the left adenovirus ITR in pAd5/Clip resulting in pAd5/Clip.sal .
- pAd5/L420-HSA.pac was digested with Avrll and Bgrlll.
- the vector fragment was ligated to a linker oligonucleotide digested with the same restriction enzymes.
- the linker was made by annealing oligos of the following sequence: PLL-1: 5'- GCC ATC CCT AGG AAG CTT GGT ACC GGT GAA TTC GCT AGC GTT AAC GGA TCC TCT AGA CGA GAT CTG G-3 ' and PLL-2: 5'- CCA GAT CTC GTC TAG AGG ATC CGT TAA CGC TAG CGA ATT CAC CGG TAC CAA GCT TCC TAG GGA TGG C-3 " .
- the annealed linkers were digested with Avrll and Bglll and separated from small ends by column purification (Qiaquick nucleotide removal kit) according to manufacturers recommendations.
- the linker was then ligated to the Avrll/Bgr ll digested pAd5/L420-HSApac fragment.
- a clone named pAdMire, was selected that had the linker incorporated and was sequenced to check the integrity of the insert.
- Adapter plasmid pAdMire enables easy insertion of complete expression cassettes.
- An adapter plasmid containing the human CMV promoter that mediates high expression levels in human cells was constructed as follows: pAd5/L420-HSA.pac was digested with Avrll and 5' protruding ends were filled in using Klenow enzyme. A second digestion with Hindlll resulted in removal of the L420 promoter sequences. The vector fragment was isolated and ligated to a PCR fragment containing the CMV promoter sequence. This PCR fragment was obtained after amplification of CMV sequences from pCMVLacI (Stratagene) with the following primers:
- CMVplus 5 ' -GATCGGTACCACTGCAGTGGTCAATATTGGCCATTAGCC-3 ' and CMVminA: 5 ' -GATCAAGCTTCCAATGCACCGTTCCCGGC-3 ' .
- the PCR fragment was first digested with Pstl (underlined in
- the adapter plasmid pCMV.LacZ was generated as follows: The plasmid pCMV.TK (EP 95-202 213) was digested with Hindlll, blunted with Klenow and dNTPs and subsequently digested with Sa l . The DNA fragment containing the CMV promoter was isolated. The plasmid pMLP.nlsLacZ (EP 95-202 213) was digested with Kpnl , blunted with T4 DNA polymerase and subsequently digested with Sail. The DNA fragment containing the LacZ gene and adjacent adenoviral sequences was isolated.
- the adapter plasmid pAd5/CLIP.LacZ was generated as follows: The E.coli LacZ gene was amplified from the plasmid pMLP.nlsLacZ (EP 95-202 213) by PCR with the primers 5 ' -GGGGTGGCCAGGGTACCTCTAGGCTTTTGCAA-3 ' and 5 ' -GGGGGGATCCATAAACAAGTTCAGAATCC-3 ' .
- the PCR reaction was performed using Ex Taq (Takara) according to the suppliers protocol at the following amplification program: 5 minutes 94°C, 1 cycle; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles; 45 seconds 94°C and 30 seconds 65°C and 2 minutes 72°C, 25 cycles; 10 minutes 72; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles, I cycle.
- the PCR product was subsequently digested with Kpnl and BamHI and the digested
- DNA fragment was ligated into Kpnl/BamHI digested pcDNA3
- the plasmid pAd/CLIP was digested with Spel .
- the large fragment containing part of the 5 ' part CMV promoter and the adenoviral sequences was isolated.
- the plasmid pcDNA3.nlsLacZ was digested with Spel and the fragment containing the 3 'part of the CMV promoter and the lacZ gene was isolated. Subsequently, the fragments were ligated, giving rise to pAd/CLIP.LacZ.
- the reconstitution of the CMV promoter was confirmed by restriction digestion.
- LacZ gene was digested from pAd/CLIP.LacZ with Kpnl and Xbal and isolated from agarose gel .
- the plasmid pAdApt was digested with Kpnl and Xbal and the large fragment was purified from agarose gel .
- the LacZ fragment and pAdApt fragment were ligated, yielding pAdApt . LacZ .
- the adapter plasmid pAd5/CLIP. Luc was generated as follows: The plasmid pCMV.Luc (EP 95-202 213) was digested with Hindlll and BamHI. The DNA fragment containing the luciferase gene was isolated. The adapter plasmid pAd/CLIP was digested with Hindlll and BamHI, and the large fragment was isolated. Next, the isolated DNA fragments were ligated, giving rise to pAd5/CLIP. Luc .
- the adapter plasmid pAd5/ULIP .LacZ . sal was generated as follows: First, the Ubiquitin C promoter (Nenoi et al . , 1996) was amplified from genomic DNA from human osteosarcoma cells (U2-OS) by a PCR reaction using the following primers:
- the adapter plasmid pAd/EF-la.LacZ. Pac is generated as follows.
- the EF-la promoter is amplified by PCR from the plasmid pEF-BOS (Mizushima and Nagata, 1990) using the primers
- pAdApt.LacZ is digested with Avrll and Acc65I, treated with Klenow enzyme and religated to generate pAd5/Nop. LacZ .
- the latter plasmid is digested with Acc65I and used in a ligation reaction with Acc65I digested PCR product containing the EF-la promoter, yielding pAd5/EF-la.LacZ .
- Example 2 Generation of producer cell lines for the production of recombinant adenoviral vectors deleted in early region 1 and early region 2A
- the producer cell lines complement for the El and E2A deletion from recombinant adenoviral vectors in trans by constitutive expression of both El and E2A genes.
- the pre-established Ad5- El transformed human embryo retinoblast cell line PER.C6 (WO 97/00326) was further equipped with E2A expression cassettes.
- the adenoviral E2A gene encodes a 72 kDa DNA Binding Protein with has a high affinity for single stranded DNA. Because of its function, constitutive expression of DBP is toxic for cells.
- the tsl25E2A mutant encodes a DBP which has a Pro—>Ser substitution of amino acid 413 (van der Vliet, 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. Temperature sensitive E2A gradually becomes functional upon temperature decrease and becomes fully functional at a temperature of 32 °C, the permissive temperature.
- pcDNA3wtE2A The complete wild-type 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) .
- the PCR was performed on a Biometra Trio Thermoblock, using the following 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
- the primer DBPpcr2 CGG AAT TCT TAA AAA TCA AAG GGG TTC TGC CGC
- 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.
- PER.C6 cells were cultured in Dulbecco ' s Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL) and lOmM MgCl 2 in a 10% C0 2 atmosphere at either 32°C, 37°C or 39°C.
- DMEM Dulbecco ' s Modified Eagle Medium
- FBS Fetal Bovine Serum
- lOmM MgCl 2 10% C0 2 atmosphere at either 32°C, 37°C or 39°C.
- DMEM Dulbecco ' s Modified Eagle Medium
- FBS Fetal Bovine Serum
- lOmM MgCl 2 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
- PER.C6 performs very well both at 32°C and 39°C, the permissive and non-permissive temperature for tsl25E2A, respectively.
- PER.C6 cells were seeded per 6 cm tissue culture dish (Greiner) in DMEM, supplemented with 10% FBS and lOmM MgCl 2 and incubated at 37°C in a 10% C0 2 atmosphere .
- the cells were transfected with 3 , 5 or 8 ⁇ g of either pcDNA3 , pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINE
- the adenovirus Ad5.dl802 is an Ad 5 derived vector deleted for the major part of the E2A coding region and does not produce functional DBP (Rice et al . , 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.
- CPE cytopathic effect
- the producer cell line preferably a suspension culture in medium devoid of any human or animal constituents.
- the cell line PER.C6tsE2A c5-9 (designated c5-9) was cultured at 39°C and 10% C0 2 in a 175 cm 2 tissue culture flask (Nunc) in DMEM, supplemented with 10% FBS and lOmM MgCl 2 .
- the cells were washed with PBS (NPBI) and the medium was replaced by 25 ml serum free suspension medium Ex-cellTM 525
- cells were seeded in a 125 ml tissue culture erlenmeyer (Corning) at a seeding density of 3 x 10 s cells per ml in a total volume of 20 ml SFM. Cells were further cultured at 125 RPM on an orbital shaker (GFL) at 39°C in a 10% C0 2 atmosphere . Cells were counted at day 1-6 in a Burker cell counter. In Figure 4, the mean growth curve from 8 cultures is shown. PER.C6tsE2A c5-9 performed well in serum free suspension culture. The maximum cell density of approximately 2 x 10 6 cells per ml is reached within 5 days of culture.
- PER.C6 cells or PER. C6tsl25E2A (c8-4) cells were cultured in Dulbecco ' s Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL) and lOmM MgCl 2 in a 10% C0 2 atmosphere at either 37°C (PER.C6) or 39°C (PER. C6tsl25E2A c8-4) .
- DMEM Dulbecco ' s Modified Eagle Medium
- FBS Fetal Bovine Serum
- lOmM MgCl 2 lOmM MgCl 2
- a total of 1 x 10 6 cells were seeded per 25cm 2 tissue culture flask (Nunc) and the cells were cultured at the respective temperatures. At the indicated time points, cells were counted.
- the PER.C6tsl25E2A cell line clone 8-4 was cultured at 39 °C and 10% C0 2 in a 25 cm 2 tissue culture flask (Nunc) in DMEM, supplemented with 10% FBS and 10 mM MgCl 2 in the absence of selection pressure (G418) .
- the cells were washed 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) .
- pcDNA3.1-tTA The tTA gene, a fusion of the tetR and VP16 genes, was removed from the plasmid pUHD 15-1 (Gossen and Bujard, 1992) by digestion using the restriction enzymes BamHI and EcoRI. First, pUHD15-l was digested with EcoRI. The linearized plasmid was treated with Klenow enzyme in the presence of dNTPs to fill in the EcoRI sticky ends. Then, the plasmid was digested with BamHI. The resulting fragment, 1025 bp in length, was purified from agarose.
- 2xl0 6 PER.C6 or PER.C6/E2A cells were seeded per 60 mm tissue culture dish (Greiner) in Dulbecco's modified essential medium (DMEM, Gibco BRL) supplemented with 10% FBS (JRH) and 10 mM MgCl 2 and incubated at 37°C in a 10% C0 2 atmosphere.
- DMEM Dulbecco's modified essential medium
- JRH FBS
- 10 mM MgCl 2 10 mM MgCl 2
- cells were transfected with 4-8 ⁇ g of pcDNA3.1-tTA plasmid DNA using the LipofectAMINE PLUSTM Reagent Kit according to the standard protocol of the supplier (Gibco BRL) .
- the cells were incubated with the LipofectAMINE PLUSTM-DNA mixture for four hours at 37°C and 10% C0 2 . Then, 2 ml of DMEM supplemented with 20% FBS and 10 mM MgCl 2 was added and cells were further incubated at 37°C and 10% C0 2 . The next day, cells were washed with PBS and incubated in fresh DMEM supplemented with 10% FBS, 10 mM MgCl 2 at either 37°C (PER.C6) or 39°C (Per.C6/E2A) in a 10% C0 2 atmosphere for three days.
- PER.C6 37°C
- Per.C6/E2A 39°C
- PER.C6 cells were incubated with DMEM supplemented with 10% FBS, 10 mM MgCl 2 and 50 ⁇ g/ml hygromycin B (GIBCO) while PER.C6/E2A cells were maintained in DMEM supplemented with 10% FBS, 10 mM MgCl 2 and 100 ⁇ g/ml hygromycin B. Colonies of cells that resisted the selection appeared within three weeks while nonresistant cells died during this period.
- a number of independent, hygromycin resistant cell colonies were picked by scraping the cells from the dish with a pipette and put into 2.5 cm 2 dishes (Greiner) for further growth in DMEM containing 10% FBS, 10 mM MgCl 2 and supplemented with 50 ⁇ g/ml (PERC.6 cells) or 100 ⁇ g/ml (PERC.6/E2A cells) hygromycin in a 10% C0 2 atmosphere and at 37°C or 39°C, respectively.
- DMEM containing 10% FBS, 10 mM MgCl 2 and supplemented with 50 ⁇ g/ml (PERC.6 cells) or 100 ⁇ g/ml (PERC.6/E2A cells) hygromycin in a 10% C0 2 atmosphere and at 37°C or 39°C, respectively.
- Adenovirus packaging cells (PER.C6) were seeded in ⁇ 25 cm 2 flasks and the next day, when they were at ⁇ 80% confluency, transfected with a mixture of DNA and lipofectamine agent (Life Techn.) as described by the manufacturer.
- viruses e.g. IG.Ad.CMV.LacZ ⁇ E2A, IG.Ad. CLIP.LacZ ⁇ E2A, IG.Ad. CLIP ⁇ E2A or IG.Ad.CLIP.Luc ⁇ E2A
- IG.Ad.CMV.LacZ ⁇ E2A IG.Ad. CLIP.LacZ ⁇ E2A
- IG.Ad.CLIP.Luc ⁇ E2A requires a complementing cell line for complementation of both El and E2A proteins in trans, as described above.
- E3 functions are not necessary for the replication, packaging and infection of the (recombinant) virus, it is also possible to delete or replace (part of) the E3 region in the El- and/or El/E2A-deleted adenoviral vector. This creates the opportunity to use larger inserts or to insert more than one gene without exceeding the maximum packagable size (approximately 105% of wt genome length) . This can be done, e.g., by deleting part of the E3 region in the pBr/Ad.Bam-rlTR clone by digestion with Xbal and re- ligation. This removes Ad5 wt sequences 28592-30470 including all known E3 coding regions.
- Another example is the precise replacement of the coding region of gpl9K in the E3 region with a polylinker allowing insertion of new sequences. This, leaves all other coding regions intact and obviates the need for a heterologous promoter since the transgene is driven by the E3 promoter and pA sequences, leaving more space for coding sequences .
- Primers 3 (5' -GAT CCC ATG GGG ATC CTT TAC TAA GTT ACA AAG CTA-3') and 4 (5' -GTC GCT GTA GTT GGA CTG G-3 ' ) were used on the same DNA to amplify Ad5 sequences from 29217 to 29476.
- the two resulting PCR fragments were ligated together by virtue of the new introduced Ncol site and subsequently digested with Xbal and Muni .
- This fragment was then ligated into the pBS .Eco-Eco/ad5 ⁇ HIII vector that was digested with Xbal (partially) and Muni generating pBS .Eco-Eco/ad5 ⁇ HIII . ⁇ gpl9K.
- pBS .Eco-Eco/ad5 ⁇ HIII. ⁇ gpl9K To allow insertion of foreign genes into the Hindlll and BamHI site, an Xbal deletion was made in pBS .Eco-Eco/ad5 ⁇ HIII. ⁇ gpl9K to remove the BamHI site in the Bluescript polylinker.
- the resulting plasmid pBS .Eco-Eco/ad5 ⁇ HIIl ⁇ gpl9K ⁇ Xbal contains unique Hindlll and BamHI sites corresponding to sequences 28733 (Hindlll) and 29218 (BamHI) in Ad5.
- ⁇ gpl9K plasmid (with or without inserted gene of interest) are used to transfer the region comprising the gene of interest into the corresponding region of pBr/Ad.Bam-rlTR, yielding construct pBr/Ad.Bam-rITR ⁇ gpl9K (with or without inserted gene of interest) .
- This construct is used as described supra to produce recombinant adenoviruses. In the viral context, expression of inserted genes is driven by the adenovirus E3 promoter.
- Recombinant viruses that are both El and E3 deleted are generated by a double homologous recombination procedure for El-replacement vectors using a plasmid-based system consisting of: a) an adapter plasmid for El replacement according to the invention, with or without insertion of a first gene of interest, b) the pWE/Ad.Aflll -EcoRI fragment, and c) the pBr/Ad.Bam-rITR ⁇ gpl9K plasmid with or without insertion of a second gene of interest .
- a plasmid-based system consisting of: a) an adapter plasmid for El replacement according to the invention, with or without insertion of a first gene of interest, b) the pWE/Ad.Aflll -EcoRI fragment, and c) the pBr/Ad.Bam-rITR ⁇ gpl9K plasmid with or without insertion of a second gene of
- Recombinant viruses that are El deleted and harbor a synthetic E4 promoter region are generated by a homologous recombination procedure as described above for El-replacement vectors using a plasmid-based system consisting of: a) an adapter plasmid for El replacement according to the invention, with or without insertion of a first gene of interest , b) pWE/Ad.AflII-rITR.tetO-E4
- IG.Ad/LacZ ⁇ EltetO-E4 is done in the appropriate complementing cells, i.e. PERC.6/tTA cells.
- Several different recombinant adenoviruses, comprising the bacterial LacZ gene (IG.Ad.AdApt .LacZ and IG.Ad.ULIP.LacZ) have been produced using this protocol (see table I) .
- Ad vectors comprising attenuated E2B or E4 in cells that do not express tTA
- a selection of recombinant Ad vectors i.e. IG .Ad .AdApt . LacZ . ⁇ E2AtetO-E4 , IG .Ad . ULIP . LacZ . ⁇ E2AtetO-E4 , IG.Ad.ULIP. LacZ. tetO-E2B, and IG.Ad.ULIP . LacZ . ⁇ E2A (control virus) , that were generated by the procedure described above, were tested for their ability to replicate in PER/E2A cells that do not express tTA. The growth of these viruses in PER/E2A/tTA cells was analyzed in parallel. Table III shows that the growth of IG.Ad.AdApt .LacZ . ⁇ E2AtetO-E4 ,
- Example 5 Biological activity of IG.Ad/ ⁇ EltetO-E4, IG.Ad/ ⁇ El ⁇ E2AtetO- E4, IG.Ad/ ⁇ El ⁇ E2AtetO-E2B, and IG.Ad/ ⁇ El ⁇ E2AtetO-E2BtetO-E4 vectors in vitro and in vivo.
- Ad vectors with conditionally disabled E2B and/or E4 genes express reduced levels of E2B and/or E4 genes, in mammalian and/or human cells
- HeLa cells ATCC CCL-2
- A549 cells are seeded at lxlO 6 cells per tissue culture plate (Greiner) in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL) in a 10% C0 2 atmosphere at 37°C.
- cells are inoculated with 0, 10, 100, 1000 or 10,000 virus particles of IG.Ad/LacZ ⁇ EltetO-E4, IG.Ad/LacZ ⁇ El ⁇ E2AtetO-E4 , IG.Ad/LacZ ⁇ El ⁇ E2Atet0- E2B or IG.Ad/LacZ ⁇ El ⁇ E2AtetO-E2BtetO-E4 per cell.
- parallel cell-cultures are inoculated with 10, 100, 1000, or 10,000 virus particles of IG.Ad/LacZ ⁇ El or IG .Ad/LacZ ⁇ El ⁇ E2A.
- cells can be either assayed for viral gene (E2, E4 and late genes) expression or for LacZ expression.
- LacZ transducing efficiency is determined as follows: Infected cells are washed twice with PBS (NPBI) and fixed for 8 minutes in 0.25% glutaraldehyde (Sigma) in PBS.
- the cells are washed twice with PBS and stained for 8 hours with X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2mM MgCl 2 (Merck), 5mM K 4 [Fe (CN) 6 ] .3H 2 0 (Merck), 5mM K 3 [Fe(CN) 6 ] (Merck) in PBS.
- X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2mM MgCl 2 (Merck), 5mM K 4 [Fe (CN) 6 ] .3H 2 0 (Merck), 5mM K 3 [Fe(CN) 6 ] (Merck) in PBS.
- IG.Ad/LacZ ⁇ El ⁇ E2AtetO-E2BtetO-E4 is injected into the tail vein of 8 weeks old C57/B16 or NOD-SCID mice. At day 7, 14, 28, and 56, two mice per group are sacrificed, the livers of these mice are isolated and fixed in formalin.
- Thin slices are cut and extensively washed in PBS. Subsequently, the slices are stained in X-gal solution (1 mg/ml X-gal in DMSO (Gibco) , 2mM MgCl 2 (Merck), 5mM K 4 [Fe (CN) 6 ] .3H 2 0 (Merck), 5mM K 3 [Fe (CN) 6 ] (Merck) in PBS. After an 8 -hour incubation, the samples are washed in PBS. The longevity of LacZ expression from the different Ad vectors will thus be assayed.
- LacZ ⁇ El ⁇ E2A vectors the expression of LacZ in C57/B16 mice is prolonged when replication-conditioned Ad vectors were used instead of the conventional IG/Ad.LacZ ⁇ El and IG/Ad . LacZ ⁇ El ⁇ E2A vectors. In contrast, the LacZ expression in the livers of immune- deficient NOD-SCID mice is expected to be stable in all cases.
- Ad vectors with conditionally disabled E4 genes express reduced levels of E4 genes in mammalian and/or human cells
- the following experiment was performed: A549 cells were seeded at lxlO 6 cells per 10 cm 2 tissue culture dish (Greiner) in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL), and incubated in a 10% C0 2 atmosphere at 37°C.
- the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLuc ⁇ El, IG.Ad/AdAptLuc ⁇ El ⁇ E2A, IG.Ad/AdAptLuc ⁇ Eltet0-E4, or IG.Ad/AdAptLuc ⁇ El ⁇ E2Atet0-E4.
- the cells were harvested at 30 h post-inoculation by lysis in 100 ⁇ l RIPA buffer (PBS + 1% NP40 + 0.5% deoxycholic acid + 0.1% SDS + protease inhibitor cocktail). After 15 min incubation on ice, the cell lysates were spun for 15 min at 14,000 rpm, 4°C in an eppendorf centrifuge. The total protein concentration in the supernatants was thereafter determined using the Bio-Rad DC Protein Assay. The (relative) amounts of the E4-orf6 (34kD) protein present in the cell extracts were determined by western blot analysis.
- the DNA was thereafter blotted onto a HybondTM N+ nylon transfer membrane (Amersham Pharmacia Biotech) and probed with a Hindlll-Nhel (484 bp) fragment of the Ad5 fiber gene that was labeled with 32 P-CTP using the Rad Prime RTS System (GIBCO) . Although some variation in the amount of vector DNA could be observed (Fig.10), it is clear that the reduced expression of E4 from IG.Ad/AdApt . Luc ⁇ EltetO-E4, and IG.Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4 cannot be explained by inefficient transduction.
- Ad vectors with conditionally disabled E4 genes express reduced levels of the E2A (DBP) gene in mammalian and/or human cells
- DBP E2A
- the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLuc ⁇ El, IG.Ad/AdAptLuc ⁇ El ⁇ E2A, IG. Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4, or IG.Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4.
- the cells were harvested as described supra and the cell extracts were analyzed for the presence of the E2A protein DBP by the western blot assay as described supra, except that the membrane was processed by incubation with the anti-DBP monoclonal antibody B6 (first antibody, 1:1000 diluted; Reich et al., 1983) and a blotting grade affinity purified Goat anti-Mouse IgG (H+L)-HRP (secondary antibody, 1:7500 diluted; Biorad) .
- the anti-DBP monoclonal antibody B6 first antibody, 1:1000 diluted; Reich et al., 1983
- Goat anti-Mouse IgG (H+L)-HRP secondary antibody, 1:7500 diluted; Biorad
- the cells were harvested at 30h post-inoculation and the relative amount of the E2B (p)TP protein present in the cell extracts was determined by Western blot analysis as described above except that this time, a mixture (1:1:1) of three antibodies against (p)TP and Pol (kind gift of P. C. van der Vliet, Utrecht, the Netherlands) was used as primary antiserum (1:500 diluted).
- the data shown in Fig.12 clearly demonstrate that the El- and El+E2A-deleted Ad vectors with conditionally disabled E4 genes produced significantly reduced amounts of the pTP protein than El or E1+E2A deleted vectors possessing wt E4.
- Ad vectors with conditionally disabled E4 genes express reduced levels of late genes in mammalian and/or human cells
- A549 cells were seeded at lxlO 6 cells per 10 cm 2 tissue culture dish in DMEM supplemented with 10% FBS, and incubated in a 10% C0 2 atmosphere at 37°C. The next day, the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLuc ⁇ El, IG.Ad/AdAptLuc ⁇ El ⁇ E2A, IG.Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4, or IG.
- the cells were harvested at 72 h post-inoculation and the relative amount of the fiber protein present in the cell extracts was determined by western blot analysis as described above except that the polyclonal E641/3 anti-knob domain of fiber (primary antibody, 1:5000 diluted; kind gift of R. Gerard, Leuven, Belgium) and the blotting grade affinity purified Goat anti-Rabbit IgG (H+L)-HRP (secondary antibody, 1:100000 diluted; Biorad) were used.
- mice are injected intravenously via the tail vein with 1E11 vp of IG.Ad/ ⁇ El, IG.Ad/ ⁇ El ⁇ E2A, IG. d/ ⁇ EltetO-E4 , IG.Ad/ ⁇ El ⁇ E2AtetO-E4, or with PBS/0.5% sucrose only.
- BALB/c, C57BL/6 and C3H mice are used. Ten mice per vector and per time-point are used.
- All vectors are suspended in PBS/0.5% sucrose, and except for modifications in the E2A and E4 regions, these vectors are genetically identical and lack a transgene and accompanying transcription elements to avoid any unintentional effect of a transgene and/or accompanying transcription elements on liver toxicity.
- the mice are sacrificed on day 3, 14, 28, 56 and 90 after injection, weighed and the livers of these mice are isolated and weighed as well. Removal of liver: liver and gall bladder are removed. Of each liver, a part of the anterior and posterior right lobe is cut-off with a clean scalpel and snap frozen in liquid nitrogen and processed for real-time PCR or Southern blot analysis (to check the transduction efficiency) .
- liver lobes (median, left and caudate) are fixed in ample buffered formalin.
- the median lobe (with biliary cyst) and left lobe are trimmed at their largest cross section for HDS staining.-
- the livers are examined histologically. Liver lesions, such as vacuolar change, apoptosis, dense nuclei, inclusions, mitotic increase, anisonucleosis, megalocytosis, and inflammation in peri-portal and sinusoidal areas are scored semi- quantitatively.
- blood is sampled for blood cell counts (erys, leucocytes, thrombocytes) and plasma is taken for biochemical measurement of ALAT, ASAT, AP, gamma-GT, ALB and TBIL. All procedures are executed according to procedures very well known to persons skilled in the art.
- CMV promoter driving a transgene is cell-type specific
- A549 cells were seeded at lxlO 6 cells per 10 cm 2 tissue culture dish in DMEM supplemented with 10% FBS, and incubated in a 10% C0 2 atmosphere at 37°C. The next day, the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLuc ⁇ El, IG. d/AdAptLuc ⁇ El ⁇ E2A, IG.Ad/AdAptLuc ⁇ Eltet0-E4, or IG. d/AdAptLuc ⁇ El ⁇ E2AtetO-E4.
- all these vectors contain the luciferase gene under the control of the CMV promoter.
- the cells were harvested by detergent- mediated lysis at 48 h post-inoculation and the luciferase activity in the cell extracts was measured and expressed in RLU (relative light units) using the Luciferase Assay System (Promega) according to the manufacturer's recommendation.
- the RLU was normalized to the total amount of protein in the cell extracts, which was measured by using the BioRad DC Protein Assay.
- the results, as shown in Fig.14 indicate that the Ad vectors possessing conditionally disabled E4 produced significantly less luciferase than the vectors possessing wt E . This implies that the CMV promoter that drives the expression of the luciferase gene was less active in vectors possessing conditionally disabled E4 than in vectors possessing wt E4.
- A549 cells were seeded in 96-well plates with a density of 10,000 cells/well in a volume of 100 ⁇ l DMEM+10% heat-inactivated FBS and incubated in a humidified C0 2 incubator set at 37°C and 10% C0 2 .
- the cells were transduced with viruses derived from PER/E2A/tTA cells transfected with the adapter plasmid pAdApt-luc in combination with pWE/Ad.
- fHI-rITR ⁇ E2AtetO-E4 or derived from PER.C6/E2A cells that were transfected with pAdApt-Luc in combination with pWE/Ad.AfHI-rITR ⁇ E2A.
- crude lysates as well as purified viruses were used.
- vp virus particles per ml were determined.
- Infections were performed for 5 time points in quadruplate per time point.
- the Multiplicities of Infections (MOI's) were 5,000 and 50,000 vp/cell in a total volume of 150 ⁇ l.
- the cells were again incubated in a humidified C0 2 incubator set at 37 °C and 10% C0 2 .
- a humidified C0 2 incubator set at 37 °C and 10% C0 2 .
- cells in plate for time point "24 hour” were washed with 100 ⁇ l PBS and lysed with 100 ⁇ l lysisbuffer (8 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 1% v/v Triton X-100 and 15% v/v glycerol) .
- the plate was frozen at -20°C until luciferase- and total protein assays could be performed.
- Luciferase experiments were performed as follows. After thawing, the plates were centrifuged for 5 min at 1500 rpm and put on ice. Luciferase expression was determined with a luminometer [EG&G Berthold] . For this, 20 ⁇ l sample was put in an appropriate tube after which the machine added 100 ⁇ l luciferase assay substrate buffer (luciferase assay substrate dissolved in 10ml luciferase assay buffer [Promega Catno. E1501] ) . Some samples were diluted in lysisbuffer because expression was too high to measure. To correct for total protein quantity in the samples, the
- CBQCA protein quantitation assay (Molecular Probes. Catno. C- 6667) was performed according the manufacturers protocol. For all samples, 5 ⁇ l was used in the assay.
- Fig.16 shows the results of the transduction of the A549 cells. These data show that the ⁇ E2A.tetO-E4 viruses (normal lines) give about a 100 fold lower expression over time as compared to the ⁇ E2A viruses (dashed lines) . There is no clear decrease in luciferase expression for each virus separately over time. After 168 hours the level of luciferase activity using these different viruses (purified or crude) is comparable to levels detected after 24 and 48 hours.
- HepG2 liver derived cells
- Transduction, luciferase and protein content determination experiments for HepG2 cells were performed according to the protocol described supra for A549 cells, with the following two exemptions.
- HepG2 cells were seeded in 96-well plates with a density of 22,500 cells/well (100 ⁇ l) and the MOI's that were used were 30 and 300 vp/cell in a total volume of 150 ⁇ l.
- Fig.17 shows the luciferase activity results obtained after the transduction of the HepG2 cells.
- the results suggest that expression over time of the ⁇ E2A.tetO-E4 (normal lines) and the ⁇ E2A viruses (dashed lines) are comparable.
- the luciferase activity derived from both viruses apparently increase over time.
- MCF-7 breast cancer derived cells
- luciferase and protein content determination experiments for MCF-7 cells were performed according to the protocol described supra for A549 cells, with the exemption that the medium used was DMEM containing 10% non-heat inactivated FBS.
- Fig.18 shows the luciferase activity results obtained after the transduction of the MCF-7 cells.
- Ad vectors possessing conditionally disabled E4 were confirmed by Southern blot analysis of the vector genome.
- the viral genomic DNA was isolated from purified IG.Ad/ ⁇ E1 ⁇ E2A and IG. d/ ⁇ El ⁇ E2AtetO- E4 virus particles. Therefore, 100 ⁇ l virus suspension containing 1.4X10 11 - 3xl0 n virus particles was mixed with 18 ⁇ l buffer (50 mM MgCl 2 , 1.2 mM CaCl 2 , and 130 mM Tris pH
- tetO This fragment corresponds to the tet operon sequence and was labeled with 32 P-CTP using the Rad Prime RTS System (GIBCO) .
- the data in Fig.19 show that only a fragment of the genomic DNA of IG.Ad/ ⁇ El ⁇ E2AtetO-E4 was labeled whereas the genomic DNA of IG.Ad/ ⁇ El ⁇ E2A was unlabeled. This indicates that only the genomic DNA of IG.Ad/ ⁇ El ⁇ E2AtetO-E4 contained the tet operon sequence.
- Serum-free suspension cultures of PER/E2A/tTA and PER.C6/tTA cells Serum-free suspension cultures of PER/E2A/tTA and PER.C6/tTA cells .
- the cell cultures were further incubated at 37°C or at 39°C.
- the standard temperature for incubating adherent cell cultures that contain a temperature sensitive E2A gene is 39°C.
- the static cultures (in flasks) were incubated at 39°C, while the dynamic cultures in roller bottles were incubated at 37°C.
- the second bio-reactor cells were cultured for 4 days after which a virus infection was performed while the cell density was approximately lxlO 6 cells per ml. For this an MOI of 70 virus particles per cell was applied with purified attenuated-E4 recombinant adenovirus. After 4 days of infection the bio-reactor run was terminated and the cells and medium were harvested.
- PER.C6/tTA The experiments to obtain a serum free suspension culture of PER.C6/tTA were similarly executed.
- 5xl0 6 PER.C6/tTA cells (clone 2C5 pn 8) were cultured in ExCell 525 medium supplemented with 4 mM L-glutamin. These cells adapted to serum free suspension medium after 2 passages after thawing.
- Culture conditions were as follows: Passaging of the cells was done after 2 or 3 days of incubation. A sample of the cell culture was used for counting and staining for determination of the cell density and viability of these cultures. The culture was then passed to a roller bottle or diluted in the same flask as it was cultured in. The culture was diluted to 2xl0 5 or 3xl0 5 viable cells per ml for an incubation period of 3 or 2 days respectively. All roller bottle (dynamic) cultures were incubated at 37°C and flushed with C0 2 .
- Two 2-liter bio-reactor runs were also performed with the PER.C6/tTA cells.
- the first bio-reactor cells were cultured for 8 days and reached a density of approximately 4xl0 6 cells per ml using standard perfusion known to persons skilled in the art.
- the second bio-reactor cells reached a density of approximately 3xl0 6 cells per ml and were subsequently infected with a concentrated batch of recombinant attenuated-E4 adenovirus that was derived from the infected roller bottle cultures described supra.
- An MOI of 70 virus particles per cell was applied, using standard perfusion methods.
- the culture was kept at 37°C. Three days after infection the virus yield in crude samples taken from the bio-reactor was measured. The concentration of this virus stock was 7xl0 10 virus particles per ml. This equals a production rate of approximately 23,000 virus particles per cell.
- Plaque purification IG.Ad/ ⁇ EltetO-E4 and IG. d/ ⁇ El ⁇ E2AtetO- E4 vectors were plaque-purified by using PER.C6/tTA and PER/E2A/tTA cells, respectively.
- PER.C6/tTA PER/E2A/tTA cells
- cells were seeded in 6-wells plates at a density of 1.5xl0 6 cells per 10 cm 2 well in DMEM + 10% FBS + 10 mM MgCl 2 . After a 4 h incubation at 37°C, 10% C0 2 (PER.C6/tTA) or 39°C, 10% C0 2 (PER/E2A/tTA) the medium was replaced by 1 ml of inoculation medium.
- the inoculation medium consisted of IG. d/ ⁇ EltetO-E4 or IG.Ad/ ⁇ El ⁇ E2AtetO-E4 vectors that had been * diluted in DMEM + 10% FBS + 10 mM MgCl 2 . Usually, 10-fold dilutions were made ranging from 10 "4 to 10 "9 .
- the cells were incubated in inoculation medium o/n at 37°C, 10% C0 2 (PER.C6/tTA) or 34°C, C0 2 (PER/E2A/tTA) .
- the inoculation medium was removed and the cells were washed with PBS and overlaid with 3 ml MEM + 10 mM MgCl 2 + 2.5% agarose + 2-5% FBS per well.
- the cells were further incubated at 37°C, 10% C0 2 (PER.C6/tTA) or 34°C, 10% C0 2 (PER/E2A/tTA) .
- PER.C6/tTA 10% C0 2
- E2A/tTA 10% C0 2
- Propagation of plaque-purified vectors the above-described resuspended plaque material containing IG. Ad/ ⁇ Eltet0-E4 or IG.Ad/ ⁇ El ⁇ E2Atet0-E4 was thawed and 100 ⁇ l of each plaque material was mixed with 900 ⁇ l of DMEM + 10% FCS + 10 mM MgCl 2 , and used to inoculate sub-confluent monolayers of PER.C6/tTA or PER/E2A/tTA cells in 2.5 cm 2 wells, respectively. Cells were incubated at 37°C, 10% C0 2 (PER.C6/tTA) or at 34°C, 10% C0 2 (PER/E2A/tTA) .
- Full CPE usually occurred after 4 to 7 days. Thereafter, the cells were scraped from the dish and harvested together with the medium. The cell/medium suspension was then frozen at -20°C. After thawing, 0.5 ml of this suspension was used to inoculate sub-confluent monolayers of .fresh cells in 80 cm 2 - culture flasks (Nunc) for further amplification. This procedure could be repeated and scaled up to large-scale vector propagations. High yields (more than 25,000 vp per cell) of progeny vectors were obtained when fresh cells were inoculated at an MOI of 50-200 vp per cell.
- PER.C6/tTA and PER/E2A/tTA cells were used to determine the infectious titer of batches of IG.Ad/ ⁇ EltetO-E4 and IG. d/ ⁇ El ⁇ E2AtetO-E4 , respectively.
- cells were seeded in 96-wells at. a density of 4xl0 4 cells per well in DMEM + 10% FBS + 10 mM MgCl 2 .
- the medium was replaced after a 4-hr incubation at 37°C, 10% C0 2 (PER.C6/tTA) or 39°, 10% C0 2 (PER/E2A/tTA) by 200 ⁇ l of serial dilutions (made in DMEM + 10% FBS + 10 mM MgCl 2 ) of IG.Ad/ ⁇ EltetO-E4 or IG.Ad/ ⁇ El ⁇ E2AtetO-E4, respectively. Cells were further incubated at 37°C, 10% C0 2 and 34°C, 10% C0 2 , respectively, and CPE was monitored every 3-4 days. After 14-16 days, the titer of the vectors was calculated from the highest dilution of vector that gave CPE.
- Example 15 Example 15
- PER/E2A/tTA (clone 1A1) cells were kept in culture for at least 100 passages in medium containing the selection drug hygromycin (100 ⁇ g/ml). Routinely, the cells were split (1:3 - 1:5) twice per week. To verify that PER/E2A/tTA cells maintain their capacity to efficiently support the propagation of El+E2A-deleted, E4-attenuated Ad vectors the propagation of such vectors was compared in PER/E2A/tTA cells that had been kept in culture for a long and short period.
- 10 "6 of the progeny vectors were made in DMEM + 10% FBS, and used to inoculate sub-confluent monolayers of A549 cells in 2.5 cm 2 wells. After a 48 h incubation at 37°C, the cells were washed twice with PBS and fixed for 8 min with 1% formaldehyde, 0.2% glutar (di) aldehyde in PBS.
- the cells were thereafter stained with X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2 mM MgCl 2 (Merck), 5 mM K 4 [Fe (CN) 6 ] .3H 2 0 (Merck), 5 mM K 3 [Fe(CN) 6 ] (Merck) in PBS).
- X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2 mM MgCl 2 (Merck), 5 mM K 4 [Fe (CN) 6 ] .3H 2 0 (Merck), 5 mM K 3 [Fe(CN) 6 ] (Merck) in PBS).
- the reaction was stopped by removal of the X-gal solution and washing the cells with PBS.
- the percentage of blue cells was thereafter determined.
- the results revealed that the progeny vectors derived from PER/E2A/tTA at passage 17 and 64 yielded very similar percentages of blue cells at
- pAd5/L420-HSA (described in published PCT patent application WO 99/55132) was digested with Avrll and Bglll.
- the vector fragment was ligated to a linker oligonucleotide digested with the same restriction enzymes.
- the linker was made by annealing oligos of the following sequence: PLL-1 (5'- GCC ' ATC CCT AGG AAG CTT GGT ACC GGT GAA TTC GCT AGC GTT AAC GGA TCC TCT AGA CGA GAT CTG G-3') and PLL-2 (5'- CCA GAT CTC GTC TAG AGG ATC CGT TAA CGC TAG CGA ATT CAC CGG TAC CAA GCT TCC TAG GGA TGG C-3'). This ligation resulted in pAdMire.
- Another batch of pAd5/L420-HSA was also digested with Avrll and 5' protruding ends were filled in using Klenow enzyme. A second digestion with Hindlll resulted in removal of the L420 promoter sequences.
- the vector fragment was isolated and ligated separately to a PCR fragment containing the CMV promoter sequence. This PCR fragment was obtained after amplification of CMV sequences from pCMVLacI (Stratagene) with the following primers: CMVplus (5' -GAT CGG TAC CAC TGC AGT GGT CAA TAT TGG CCA TTA GCC-3' ) and CMVminA (5' -GAT CAA GCT TCC AAT GCA CCG TTC CCG GC-3').
- the PCR fragment was first digested with Pstl after which the 3' -protruding ends were removed by treatment with T4 DNA polymerase. Then the DNA was digested with Hindlll and ligated into the Avrll/Hindlll digested pAd5/L420-HSA vector. The resulting plasmid was named pAd5/CMV-HSA. This plasmid was then digested with Hindlll and BamHI and the vector fragment was isolated and ligated to the Hindlll/Bglll polylinker sequence obtained after digestion of pAdMire. The resulting plasmid was named pAdApt .
- EPO- START 5' AAA AAG GAT CCG CCA CCA TGG GGG TGC ACG AAT GTC CTG CCT G-3' and EPO-STOP: 5'AAA AAG GAT CCT CAT CTG TCC CCT GTC CTG CAG GCC TC-3' (Cambridge Bioscience Ltd) in a PCR on a human adult liver cDNA library.
- the amplified fragment was cloned into pUC18 linearized with BamHI. Sequence was checked by double stranded sequencing.
- the full length human EPO cDNA containing a perfect Kozak sequence for proper translation was removed from the pUC18 backbone after a BamHI digestion.
- the cDNA insert was purified over agarose gel and ligated into pAdApt which was also digested with BamHI, subsequently dephosphorylated at the 5' and 3' insertion sites using SAP and also purified over agarose gel to remove the short BamHI- BamHI linker sequence.
- the obtained circular plasmid was checked with Kpnl, Ddel and Ncol restriction digestions that all gave the right size bands. Furthermore, the orientation and sequence were confirmed by double stranded sequencing.
- the obtained plasmid with the human EPO cDNA in the correct orientation was named pAdApt.EPO and was further digested with Hindlll and Xbal restriction enzymes.
- This EPO insert was isolated and ligated to a Hindlll/Xbal digested pIPspAdapt ⁇ plasmid (described in WO 99/64582) .
- the resulting plasmid was named pAd/pIPspAdapt-hEPO.
- DNA/Lipofectamine mixture was left at room temperature for 30 min, followed by the addition of 1.95 ml DMEM. The latter mixtures were then added (30 ⁇ l per well) to the PER/E2A/tTA cells, after removal of the medium in which the cells were seeded. After 2 hours incubation in a humidified C0 2 incubator (39°C, 10% C0 2 ) , 170 ⁇ l culture medium was added to each well and the plates were returned to the humidified C0 2 incubator (39°C, 10% C0 2 ) . At day 3, the supernatant in each well was replaced with 200 ⁇ l culture medium. The plates were then placed again in another humidified C0 2 incubator (34°C, 10% C0 2 ) .
- PER/E2A/tTA cell cultures (1A1, 1C1, 1C3, 2B3, 2C3 and 2D5) were harvested and diluted in culture medium (DMEM+10% FBS and 10 mM MgCl 2 ) to a density of 22,500 cells per 100 ⁇ l . Subsequently, two 96-well-tissue culture plates for each clone were used to. seed 100 ⁇ l of the cell suspensions per well. At day 2, three different DNA mixes were prepared for each PER/E2A/tTA clone.
- culture medium DMEM+10% FBS and 10 mM MgCl 2
- a linearized adapter molecule (being either pAd/pIPspAdapt- eGFP, or pAd/pIPspAdapt-lacZ or pAd/Adapt-ceNOS) was mixed with 4 ⁇ g of Pad linearized pWE/Ad.
- tetO-E4 in 100 ⁇ l DMEM.
- 100 ⁇ l Lipofectamine mix (25.6 ⁇ l Lipofectamine (Life Techn.) + 74.4 ⁇ l DMEM) was added. This DNA/Lipofectamine mixture was left at room temperature for 30 min, after which 1.3 ml DMEM was added.
- E3 deleted versions of the vectors carrying a tetO-E4 attenuation the following cloning steps were performed.
- pBr/Ad.Bam-rITR ⁇ E2AtetO-E4 was propagated in E. coli strain DM1 (dam ' , dcm ⁇ ) (Life Techn.).
- the purified plasmid was digested with Xbal, hereby removing the 1.88 kb Xbal-Xbal insert, and subsequently religated.
- the following sequences were deleted: 191 bp of the E3-6.7K protein, the E3-19K glycoprotein, the E3-ADP (10.5K protein) , RIDalpha (E3-10.4K protein) , RIDbeta (E3- 14.6K protein) and the first 21 bp of the E3-14.7K protein (for sequences, see Genbank accession number X03002) .
- the resulting plasmid was named pBr/Ad.
- the plasmid pWE/Ad.AfHI-rITR ⁇ E2A was first digested with BamHI and then partially digested with Pad, yielding amongst others a 26.2 kb fragment.
- the 11 kb BamHI-PacI fragment from and the 26.2 kb fragment were ligated, yielding cosmid pWE/Ad.AfHI-rITR ⁇ E2AtetO-E4. ⁇ E3(XbaI).
- This cosmid contains sequences identical to that of pWE/Ad.AflII-rITR ⁇ E2AtetO-E4 but with the deletion of the Xbal-Xbal fragment.
- pWE/Ad. fHI-rITR ⁇ E2AtetO-E4. ⁇ E3 (Xbal) was used in the production of adenoviruses with El, E2A and E3 deletions and E4 attenuations in 96 well plates.
- PER/E2A/tTA clone 1C3 was harvested and diluted with culture medium (DMEM+10% FBS and 10 mM MgCl 2 ) to a density of 22,500 cells per 100 ⁇ l, followed by seeding 100 ⁇ 1 per well in 96-well-tissue culture plates.
- culture medium DMEM+10% FBS and 10 mM MgCl 2
- linearized adapter molecules pAd/pIPspAdapt-ceNOS, pAd/pIPspAdapt-eGFP, pAd/pIPspAdapt-hEPO, pAd/pIPspAdapt- hIL3, pAd/pIPspAdapt-lacZ and pAd/pIPspAdapt-luciferase were used for transfection in combination with 2 different Pad linearized helper cosmids: pWE/Ad.AfHI-rITR ⁇ E2AtetO-E4 and pWE/Ad.AflII-rITR ⁇ E2AtetO-E4. ⁇ E3 (Xbal) .
- the DNA transfection procedure was identical to that described in Example (above) describing transfections in a subset of PER/E2A/tTA clones.
- CPE formation was monitored during a period of three weeks after transfection.
- Fig.22 shows the percentage of CPE positive wells in a comparison between pWE/Ad. f111-rITR ⁇ E2AtetO-E4 and pWE/Ad.AfHI-rITR ⁇ E2AtetO-E4. ⁇ E3 (Xbal) transfections .
- Fig.23 shows the percentage of virus propagation (CPE positive wells) in 96-wells plates seeded with fresh PER/E2A/tTA cells. This was scored after infection with the supernatants from the freeze/thawed transfected cells as shown in Fig.22.
- Fig.24 shows the percentage of functional viruses that produce either human IL-3, LacZ or Luciferase in the 96-wells setting.
- microarray experiments are performed. This allows the measurement of mRNA expression of thousands of genes simultaneously and the effect a particular adenoviral vectors has on these gene expressions .
- expression profiles are determined for adenoviral vectors with different deletions and attenuations by hybridizing the labeled probes to microarrays (reviewed in Marshall and Hodgson 1998; Ramsay 1998).
- the fluorescent signals of both Cy3 and Cy5 are determined using a microarray scanner and converted using controls for hybridization and cDNA labeling and generation to normalized fluorescent values.
- the Cy3 and Cy5 values are then compared which results in relative data showing the difference in response of cells to infection with different types of adenoviral vectors including tetO-E4 attenuated and non tetO-E4 attenuated vectors.
- the in vivo duration of N0S3 transgene expression after aerosol gene transfer of rat lungs with the recombinant adenovirus IG.Ad/ ⁇ El ⁇ E2AtetO-E4-ceNOS was assessed. Twelve male Wistar rats (body weight 300 ⁇ 350 g) were aerosolized via a silastic catheter into the trachea with IG.Ad/ ⁇ El ⁇ E2AtetO-E4-ceNOS recombinant adenovirus (300 ⁇ l physiologic salt solution containing 3xl0 9 plaque forming units over 60 minutes) . After viral delivery, the catheter was removed from the trachea and animals were extubated. No side effects were observed during aerosol delivery or following extubation.
- Nitric Oxide (NO) levels in exhaled air were measured using standard chemiluminescence (Sievers 280 - NO analyzer NOATM) .
- ppm parts per million
- the minimal detectable NO concentration was 1 ppb.
- Figure 1 (A) Expression of DBP, Penton and Fiber.
- A549 cells were infected with a multiplicity of infection (m.o.i.) of either 0, 100, 1,000 or 10,000 vp/cell of IG.Ad/CLIP or IG.Ad. CLIP ⁇ E2A. Seventy-two hours post infection, cell extracts were prepared and equal amounts of whole cell extract were fractionated by SDS-PAGE in 10% gels. The proteins were visualized with the ⁇ DBP monoclonal B6, the polyclonal ⁇ -Penton base Ad2-Pb571 or the polyclonal ⁇ - knob domain of fiber E641/3, using an ECL detection system. Cells infected with IG.Ad. CLIP express both E2A encoded DBP, Penton base and Fiber proteins.
- the proteins co-migrate with the respective proteins in the positive control (lane P, extract from PER.C6 cells infected with IG.Ad. CLIP harvested at starting CPE) .
- no DBP, penton-base or fiber was detected in the non-infected A549 cells or cells infected with IG.Ad.CLIP ⁇ E2A.
- the blot as shown in A, was stripped and used to visualize the E4-orf6 and pTP/TP proteins using the method described above but now by using a monoclonal antibody against E4-orf6 or a polyclonal anti-pTP/TP antiserum, respectively. These proteins co-migrate with the respective proteins in the positive control (P) .
- the results show that pTP/TP as well as E4-orf6 are still produced from the E1/E2A deleted Ad vector. This indicates that the deletion of the E2A gene did not eliminate the residual expression of the E2B and E4 genes.
- PER.C6 cells were cultured in Dulbecco ' s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL) and lOmM MgCl 2 in a 10% C0 2 atmosphere at either 32°C, 37°C or 39°C.
- FBS Fetal Bovine Serum
- lOmM MgCl 2 Fetal Bovine Serum
- 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 39°C.
- cells were counted.
- the growth rate and the final cell density of the PER.C6 culture at 39°C are comparable to that at 37°C.
- the growth rate and final density of the PER.C6 culture at 32°C were slightly reduced as compared to that at 37°C or 39°C.
- c PER.C6 cells were seeded at a density
- Figure 3 DBP levels in PER.C6 cells transfected with pcDNA3, pcDNA3wtE2A or pcDNA3tsl25E2A.
- Equal amounts of whole-cell extract were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes and DBP protein was visualized using the ⁇ DBP monoclonal B6 in an ECL detection system. All of the cell lines derived from the pcDNA3tsl25E2A transfection express the 72-kDa E2A-encoded DBP protein (left panel, lanes 4-14; middle panel, lanes 1-13; right panel, lanes 1- 12) . In contrast, the only cell line derived from the pcDNAwtE2A transfection did not express the DBP protein (left panel, lane 2) .
- 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-cellTM medium.
- FIG. 5 Growth curve PER.C6 and PER.C6tsE2A.
- PER.C6 cells or PER.C6tsl25E2A (c8-4) cells were cultured at 37°C or 39°C, respectively.
- a total of 1 x 10 6 cells was seeded per 25cm 2 tissue culture flask.
- cells were counted.
- the growth of PER.C6 cells at 37°C is comparable to the growth of PER.C6tsl25E2A c8-4 at 39°C. This shows that constitutive overexpression of tsl25E2A has no adverse effect on the growth of cells at the nonpermissive temperature of 39°C.
- FIG. 6 Stability of PER.C6tsl25E2A.
- the PER. C6tsl25E2A cell line clone 8-4 was cultured at 39°C in medium without G418. Equal amounts of whole-cell extract from different passage numbers were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes and DBP protein was visualized using the ⁇ DBP monoclonal B6 in an ECL detection system.
- the expression of tsl25E2A encoded DBP is stable for at least 16 passages, which is equivalent to approximately 40 cell doublings. No decrease in DBP levels were observed during this culture period, indicating that the expression of tsl25E2A is stable, even in the absence of G418 selection pressure .
- FIG. 7 tTA activity in hygromycin resistent PER.C6/tTA (A) and PER/E2A/tTA (B) cells.
- Sixteen independent hygromycin resistent PER.C6/tTA cell colonies and 23 independent hygromycin resistent PER/E2A/tTA cell colonies were grown in 10 cm 2 wells to sub-confluency and transfected with 2 ⁇ g of pUHC 13-3 (a plasmid that contains the reporter gene luciferase under the control of the 7xtetO promoter) .
- pUHC 13-3 a plasmid that contains the reporter gene luciferase under the control of the 7xtetO promoter
- One half of the cultures was maintained in medium containing doxycycline to inhibit the activity of tTA.
- Cells were harvested at 48 hours after transfection and luciferase activity was measured. The luciferase activity is indicated in relative light units (RLU) per
- E4-34 kDa protein and a Southern blot to visualize the cell-associated viral DNA.
- A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLuc ⁇ El (dEl), IG.Ad/AdAptLuc ⁇ El ⁇ E2A (dE2A) , IG. d/AdAptLuc ⁇ EltetO-E4 (dEl . tetO-E4 ) , or IG.Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4 (dEl . dE2A. tetO-E4 ) .
- the cells were harvested and the relative amount of E4-orf6 protein in each sample was determined by
- the infections (described for Fig.8) for each analysis were done in triplicate and analyzed by western blotting.
- A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLuc ⁇ El (dEl), IG.Ad/AdAptLuc ⁇ El ⁇ E2A (dE2A) , IG.Ad/AdAptLuc ⁇ EltetO-E4 (dEl . tetO-E4 ) , or IG. Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4 (dEl . dE2A. tetO-E4 ) . All infections were performed in triplicate.
- the cells were harvested and the relative amount of DBP protein in each sample was determined by Western blotting using the anti-DBP monoclonal antibody B6 (first antibody, 1:1000 diluted; Reich et al., 1983).
- first antibody 1:1000 diluted; Reich et al., 1983.
- one culture was analyzed that not been infected with any vector (mock) .
- a positive control a lysate of cells in which an Ad vector had undergone replication, had been taken.
- A549 cells were inoculated with 1000 vp per cell using El- deleted Ad vectors (dEl), El+E2A-deleted Ad vectors (dE2A) , El-deleted and E4-attenuated (dEl . tetO-E4 ) , or El+E2A-deleted and E4-attenuated (dEl.dE2A. tetO-E4) .
- the cells were harvested and the relative amount of the E2B encoded protein pTP in each sample was determined by Western blotting using a mixture (1:1:1) of three antibodies against (p)TP and Pol.
- A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLuc ⁇ El (dEl), IG. d/AdAptLuc ⁇ El ⁇ E2A (dE2A) , IG.Ad/AdAptLuc ⁇ EltetO-E4 (dEl . tetO-E4 ) , or IG. d/AdAptLuc ⁇ El ⁇ E2AtetO-E4 (dEl . dE2A. tetO-E4 ) . All infections were performed in triplicate.
- A549 cells were inoculated with IG.Ad/AdAptLuc ⁇ El (dEl), IG.Ad/AdAptLuc ⁇ El ⁇ E2A (dEl.dE2A), IG. d/AdAptLuc ⁇ EltetO-E4 (dEl.tetO-E4) , or IG.Ad/AdAptLuc ⁇ El ⁇ E2AtetO-E4
- E2AtetO-E4 (dE2A. tetO-E4 ) . All infections were performed in triplicate and using the indicated multiplicity of infection (MOI) . The cells were harvested by detergent-mediated lysis at 48h post-inoculation and the luciferase activity in the cell extracts was measured and expressed in relative light units (RLU) per ⁇ g protein present in the cell extracts.
- MOI multiplicity of infection
- Fig.19 Viral genomic DNA was isolated from equal numbers of purified El+E2A-deleted (Ad.dE2A) and El+E2A-deleted, E4-attenuated (Ad.dE2A. tetO-E4) Ad vector particles and cut with Pstl.
- Ad vector particles Ad vector particles and cut with Pstl.
- the plasmid pWE/Ad.Af111-rITR. tetO-E4 was digested with Pad and Pstl
- the plasmid pNEB-PaSe. tetO was digested with Hindlll.
- the DNA samples were run in a 1% agarose gel, blotted onto a membrane and probed with a 32 P- " labeled 313 bp Hindlll/Ncol fragment of pNEB-PaSe . tetO.
- Fig.20 Percentage of CPE positive wells, based on the results of the first transfection round. CPE was scored three weeks after transfection.
- Fig.21 Percentage of CPE positive wells in the second transfection round, scored three weeks after transfection in six different PER/E2A/tTA clones transfected with either pAd/pIPspAdapt- eGFP, or pAd/pIPspAdapt-lacZ or pAd/Adapt-ceNOS . All three different transfections were in addition to pWE/Ad.Af111-rITR ⁇ E2A.tetO-E4.
- PER/E2A/tTA cells transfected with lacZ adapter plasmid in combination with either pWE/Ad .
- Table I Generation of E2B and E4 attenuated recombinant adenoviral vectors.
- This table shows a selection of E2B and E4 attenuated Ad vectors that have been generated by cotransfection of the indicated plasmid DNAs into the indicated cells.
- a cytopathic effect (CPE) typical for adenovirus replication (the appearance of rounded cells and so-called comets in the cell monolayer) is usually detected at 8-14 days after transfection. Transfection of only one of the plasmids did not result in CPE (not shown) .
- Table II A and B Growth of E2B and E4 attenuated recombinant adenoviral vectors in PER.C6/E2A and PER/E2A/tTA cells.
- the indicated viruses were diluted as indicated (from 10° - 10 ⁇ 7 ) and used to inoculate subconfluent monolayers of the indicated cells grown in 10 cm 2 wells. Parallel cultures of the cells were not incubated with virus (mock) . The cells were incubated at 34 °C. CPE was scored at 4 (A) and 5 (B) days after inoculation. 0 means no CPE; 0-1 means sporadic comet formation; 1 means several comets present in the culture, 25% of the cells show CPE; 2 means 50% of the cells shows CPE ; 3 means 75% of the cells show CPE; 4 means 100% of the cells show CPE.
- adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J. Virol. 64;2103-2109.
- helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J. Virol. 71 ; 3293-3298.
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WO2007134325A2 (en) * | 2006-05-15 | 2007-11-22 | Introgen Therapeutics, Inc. | Methods and compositions for protein production using adenoviral vectors |
WO2008060357A2 (en) * | 2006-09-29 | 2008-05-22 | Canji, Inc. | Methods of polypeptide production |
EP3945094A1 (en) * | 2020-07-31 | 2022-02-02 | Heinrich-Pette-Institut | Replication-deficient adenovirus |
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WO1997000326A1 (en) * | 1995-06-15 | 1997-01-03 | Introgene B.V. | Packaging systems for human recombinant adenovirus to be used in gene therapy |
WO1997045550A2 (en) * | 1996-05-31 | 1997-12-04 | Baxter International Inc. | Mini-adenoviral vector |
WO1998021350A1 (en) * | 1996-11-13 | 1998-05-22 | Board Of Regents, The University Of Texas System | Diminishing viral gene expression by promoter replacement |
WO1998022609A1 (en) * | 1996-11-20 | 1998-05-28 | Genzyme Corporation | Chimeric adenoviral vectors |
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WO1997000326A1 (en) * | 1995-06-15 | 1997-01-03 | Introgene B.V. | Packaging systems for human recombinant adenovirus to be used in gene therapy |
WO1997045550A2 (en) * | 1996-05-31 | 1997-12-04 | Baxter International Inc. | Mini-adenoviral vector |
WO1998021350A1 (en) * | 1996-11-13 | 1998-05-22 | Board Of Regents, The University Of Texas System | Diminishing viral gene expression by promoter replacement |
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JI L. ET AL.: "Reduced toxicity, attenuated immunogenicity and efficient mediation of human p53 gene expression in vivo by an adenovirus vector with dleted E1-E3 and inactivated E4 by GAL4-TATA promoter replacement.", GENE THERAPY, vol. 6, March 1999 (1999-03-01), pages 393 - 402, XP000876528 * |
RITTNER K. ET AL.: "Conditional repression of the E2 transcription unit in E1-E3-deleted adenovirus vectors is correleated with a strong reduction in viral DNA replication and late gene expression in vitro.", JOURNAL OF VIROLOGY, vol. 71, no. 4, April 1997 (1997-04-01), pages 3307 - 331, XP002130029 * |
Cited By (6)
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WO2007134325A2 (en) * | 2006-05-15 | 2007-11-22 | Introgen Therapeutics, Inc. | Methods and compositions for protein production using adenoviral vectors |
WO2007134325A3 (en) * | 2006-05-15 | 2008-04-03 | Introgen Therapeutics Inc | Methods and compositions for protein production using adenoviral vectors |
WO2008060357A2 (en) * | 2006-09-29 | 2008-05-22 | Canji, Inc. | Methods of polypeptide production |
WO2008060357A3 (en) * | 2006-09-29 | 2008-10-16 | Canji Inc | Methods of polypeptide production |
EP3945094A1 (en) * | 2020-07-31 | 2022-02-02 | Heinrich-Pette-Institut | Replication-deficient adenovirus |
WO2022023535A1 (en) * | 2020-07-31 | 2022-02-03 | Heinrich-Pette-Institut Leibniz-Institut für experimentelle Virologie | Replication-deficient adenovirus |
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