AU7560500A

AU7560500A - Modified adenoviral vectors for use in gene therapy

Info

Publication number: AU7560500A
Application number: AU75605/00A
Authority: AU
Inventors: Dirk Jan Elbertus Opstelten; Govert Johan Schouten; Ronald Vogels
Original assignee: Introgene BV
Current assignee: Janssen Vaccines and Prevention BV
Priority date: 1999-09-10
Filing date: 2000-09-11
Publication date: 2001-04-17
Also published as: WO2001020014A1; CA2384439A1

Description

WO01/20014 PCT/NL00/00640 Title: MODIFIED ADENOVIRAL VECTORS FOR USE IN GENE THERAPY 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 5 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. The development of human gene therapy for the -treatment of 10 inherited and acquired disorders requires gene transfer vectors capable of safe and effective delivery and expression of therapeutic genes into target cells. The actual method to introduce and express the genetic material into the target cells of a patient is a key component in every gene therapy 15 protocol. Several different gene transfer systems are currently being employed to introduce therapeutic genes into somatic cells. Non-viral gene systems for in vivo delivery into target cells include a variety of DNA-mediated methods, like direct 20 injection of naked DNA and particle bombardment. To overcome the limitations of these DNA-based delivery methods (low transfer efficiency, and cell toxicity), alternative systems have been developed utilizing the entrapment of the DNA in vesicles (like liposomes), or binding of the DNA to synthetic 25 conjugates (like e.g. transferrin-polylysine). Such 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 30 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gene-transfer efficiency of viruses has led to the development of viral vectors in which part of the viral genome has been replaced by a transgene to be introduced into eukaryotic cells.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) 35 and adeno-associated viruses. Each of these viral delivery WO01/20014 PCT/NL00/00640 2 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 5 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 10 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. 15 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. The presentation of foreign antigens usually elicits a response of the immune system; immune responses 20 directed against the gene therapy vehicle and/or the transgene product may lead to inflammation, elimination of the transduced cells, and difficulties with re-administration of the vector due to neutralizing activity against the vehicle. As a consequence, the clinical application of gene 25 therapy vectors can be impeded by the potent host immune response to the vector, which limits the duration of its effects. Immune effectors have been identified as cytotoxic T lymphocytes (CTLs), which destroy vector-transduced cells, as well as B cells7- which secrete neutralizing antibodies that 30 can block repeated gene transfer. 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 35 fragments (i.e. peptides) of foreign antigens that are presented at the cell surface in association with class I WO01/20014 PCT/NL00/00640 3 molecules of the major histocompatibility complex (MHC; for a recent review see Ploegh et al., 1998). The majority of these peptides are generated in the cytosol of virus-infected cells by degradation of poly-ubiquitinated viral proteins. The 5 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. In the ER, MHC class I heavy chains assemble with P2 microglobulin and peptide into stable hetero-trimeric MHC 10 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 anti viral defense. In the first generation of adenoviral vectors for gene 15 therapy, the El region was replaced by foreign genetic information; e.g., the therapeutic gene. The absence of El renders the recombinant virus replication defective. Because El has been reported to trigger the transcription of other Ad genes, it was previously thought that El-deleted vectors 20 would not express any Ad genes. However, it has been shown by others and us that early (e.g. E2A, E2B and E4) and late (e.g. fiber, hexon and penton-base) genes are still residually expressed from such vectors. This residual Ad gene expression is due to background replication of the 25 recombinant viral genome and the background activity of the promoters driving the transcription of the respective Ad genes (Yang et al., 1994; Lusky et al., 1998). This means that the delivery of a therapeutic gene into target cells using El-deleted Ad vectors results in expression of the 30 therapeutic gene as well as of the viral genes. Eventually, this will lead to the presentation of viral peptides by MHC class I complexes followed by a cytotoxic immune response against the transduced cells. It has been shown that CTLs directed against the transgene product as well as against the 35 Ad gene products are activated following administration of the vector into immune-competent animals (Song et al., 1997; WO01/20014 PCT/NL00/00640 4 Yang et al., 1996). The activated CTLs subsequently eradicate the transduced cells from the recipient. In order to reduce the residual expression of viral genes from recombinant Ad vectors, we have generated Ad 5 vectors that are deleted for the El and E2A region. To complement the deletions of El and E2A, we have generated an El + E2A complementing cell line for the manufacturing of high titer E1/E2A deleted recombinant Ad vector batches, as described herein. Due to the toxicity of the E2A encoded DNA io binding protein (DBP), it is difficult to generate a cell line that constitutively expresses E2A. Therefore, we have made use of a mutant E2A gene, derived from H5ts125, that encodes a temperature sensitive (ts) DBP (van der Vliet, 1975). The tsDBP is functionally active at the permissive 15 temperature (32 0 C), whereas it is nonactive at the nonpermissive temperature (390).- In addition, tsDBP is not toxic at the nonpermissive temperature. Thus, we established a new cell line, designated PER/E2A, that constitutively expresses high levels of the tsE2A gene. This cell line can 20 easily be cultured at the nonpermissive temperature. When 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 25 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 30 lead to the formation of reverted viruses that have recaptured the El and/or E2A genes. 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). However, these vectors 35 still expressed significant amounts of the E4, e.g. E4-orf6, and E2B genes, e.g., pTP (Figure IB). Transcription of E4 may WO 01/20014 PCT/NL00/00640 5 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). Hence, cells infected by E1/E2A deleted vectors still 5 produce and present non-self antigens, when delivered to humans and may be eradicated from the recipient by the immune system. In order to solve such problems the present invention provides modifications to E2B and/or E4 regions or regions controlling the E2B and/or E4 regions in adenoviral vectors 10 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 15 which have been identified in infected cells. Genetic studies have shown that 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 20 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, 25 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 30 (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 35 encoding these proteins (Sandler and Ketner, 1991). Moreover, they maintain the nuclear stability of unprocessed pre-mRNAs WO01/20014 PCT/NL00/00640 6 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. In addition, the E4-orf6 5 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 10 transcription and RNA processing occur (Pombo et al., 1994). Finally, both the E4-orf6 and E4-orf3 encoded proteins are required for Ad DNA synthesis. Proteins encoded by E4 also interact with cellular proteins and antagonize cellular processes. For example, E4 s15 protein products are involved in controlling the cellular transcription factor E2F as well as the phosphorylation of cellular (and viral) proteins. Moreover, both 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, 20 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). As a result, the E4-orf6 protein may prevent the induction of apoptosis by p53. 25 Taken together, disrupting or deleting the E4 function from recombinant 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. In addition, vectors from which the E4 function is impaired 30 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. It is important to note, however, that the impairment of the E4 function within the Ad vector backbone can influence 35 the activity of the promoter that drives the expression of the transgene. For example, it has been reported that the WO01/20014 PCT/NL00/00640 7 activity of either the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) promoter is down-regulated when the E4 region is largely or completely deleted from the Ad vector (Armentano et al., 1997; Brough et al., 1997; Dedieu et al., 5 1997). Thus, although the transduced cells and the vector DNA may persist, the expression of the transgene from the CMV or the RSV promoter is only transient when an E4-specific factor(s) is absent. The underlying mechanism causing the silencing of these promoters is unclear, but a recent study 10 point to the requirement of E4-orf3 for long-term expression from the CMV promoter (Armentano, 1999). The same study also showed that a truncated form of the CMV promoter could persistently drive expression in the absence of E4. It is yet unknown whether promoters from genes of mammals (mammalian 15 promoters) are influenced by the E4 region when present in the context of a recombinant Ad vector. It is therefore highly valuable to determine whether mammalian promoters, such as the elongation factor-1 alpha (EF-la) or the ubiquitin-C (UbC) promoter, can mediate persistent expression 20 of the transgene from an Ad vector that does not produce E4. 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 25 are transcribed from the same promoter region, designated the E2 promoter. At early time points after infection, E2 is transcribed from the E2-early promoter located at 76 map units. At intermediate times after infection, the transcription switches to another promoter, the E2-late 30 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 35 is less well understood. This promoter consists of a TATA- WO01/20014 PCT/NL00/00640 8 like sequence, SP-1 binding sites and a CAAT box. The E2-late promoter is not regulated by proteins of the ElA region. The E2A region encodes the 72kDa single stranded DNA binding protein (DBP). It plays a pivotal role in both the 5 initiation and elongation of Ad DNA replication (reviewed by van der Vliet, 1995 and references therein). Briefly, 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, 10 facilitates binding of the pTP/pol complex (see below) to the core region of the ITR. Secondly, DBP stimulates the NF1 dependent formation of pTP-dCMP, which forms the DNA replication initiation complex. Thirdly, DBP facilitates Ad DNA template unwinding by destabilization of the DNA helix in 15 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. 20 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 25 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). Transcription of E2 gives rise to two E2B-specific mRNAs that are derived from differential splicing. They encode two 30 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. Next, the ITR partially unwinds and the replication-initiation reaction is 35 primed, i.e. a pTP-dCMP coupling takes place that is catalyzed by pol. This is followed by synthesis of a pTP-CAT WO01/20014 PCT/NL00/00640 9 intermediate at G4-T5-A6 of the ITR. The pTP-trinucleotide intermediate jumps back to the Gl-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 5 enhanced by DBP, as discussed above, and progeny DNA accumulates. Finally, 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 10 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 15 incoming pTP/pol heterodimer at the core region of the ITR during the initiation of DNA replication in the next lytic infection cycle. The deletion approach to knock out the viral gene 20 functions from the recombinant Ad vector is difficult to apply to E2B and E4 for the following reasons. First, since both E4 and E2B encoded proteins are pivotal for the lytic infection cycle of Ad, the production of recombinant Ad vectors that lack these regions would require a complementing 25 cell line that is equipped with expression vectors that ectopically express, in addition to El and/or E2A, at least E4-orf6 and/or E4-orf3 as well as pTP and pol. Although the generation of such cell lines may be possible, these cell lines will normally be unstable. In addition, some of these 30 viral gene products, e.g., 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 35 not support manufacturing of high titer batches of the WO01/20014 PCT/NL00/00640 10 respective recombinant Ad vectors (Lusky et al., 1998 and references therein). Secondly, the approach to delete viral gene functions from the recombinant Ad vectors is difficult to apply to E2B. 5 Although it is possible to generate cell lines that constitutively express pTP and pol (Amalfitano et al., 1997), it is not possible to entirely delete the pTP and pol coding sequences from the vector genome. This is due to the fact that other viral regulatory elements and genes are present in 10 this area of the viral genome. This area includes the second and third tripartite leader sequences, the i-leader, portions of the major-late promoter intronic sequences and the IVa2 gene (Amalfitano et al., 1997). As a consequence, only partial deletions in the pTP and pol coding sequences can be 15 made. This yields a substantial sequence overlap between the remaining sequences in the recombinant adenoviral vector and the pTP and pol sequences in the complementing cell line. This, in turn, can lead to homologous recombination and reversion of the pTP and pol deleted phenotype during virus 20 replication. Therefore, the deletion approach is not suitable for the production of a homogeneous population of E2B crippled Ad vectors. Thirdly, deletion of the entire E4 transcription unit is feasible but it seems to impair the expression of the 25 L5 region encoding the fiber protein (Brough et al., 1996). This causes a severe reduction in virus yields when an E4 deleted vector is grown in E4 complementing cells. This defect can be partially solved by introducing a spacer sequence, e.g., an expression cassette, in place of the 30 deleted E4 sequences (GenVec, US patent 5,851,806). Fourthly, it should be noted that recombinant Ad vectors that are deleted of some or even all viral coding sequences (so-called gutless or minimal vectors) can only be propagated using a helper Ad that supports replication and 35 packaging of such vectors by providing all the necessary proteins in trans (Fisher et al., 1996; Hardy et al., 1997; WO01/20014 PCT/NL00/00640 11 Kochanek et al., 1996; Kumar-Singh and Chamberlain, 1996). Generally, the packaging sequence of such a helper virus is flanked by lox sites which are targets for the CRE recombinase. Hence, the packaging signal is excised from the s 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, however, has serious limitations with respect to the efficiency of excision of the packaging signal 10 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 15is 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 20 (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 25 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) 30 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 35 generating a recombinant adenoviral vector lacking ElA and preferably ElB sequences, but having at least the E2B and/or WO01/20014 PCT/NL00/00640 12 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. 5 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 10 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 s15 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. An additional advantage of at least some E4 modifications according to the invention, is that concurrently E2B 20 expression is at least in part reduced in said gene delivery vehicle. Thus expression of E2B can be reduced directly by modifying E2B sequences or indirect by modifying E4 sequences or by both type of modifications. Similarly, expression of E4 gene products can be reduced by modification of E4 sequences 25 or by modification of E2B sequences or both. As disclosed herein, 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 30 highly desired. Attenuation is preferably achieved through at least one mutation in at least an E2B and/or E4 promoter. Thus 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 35 attenuated through a mutation therein. In the alternative or in combination with the above the invention provides in one WO01/20014 PCT/NL00/00640 13 embodiment a method according as disclosed herein before, wherein 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 5 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 10 similar transactivation signal. Typically a complementation cell would then be able to provide said signal through e.g. an expression cassette introduced therein. As described below it is preferred that the vector also lacks a functional E2A region, which can be elegantly provided by a 15 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 ElB 55kD. Thus the invention also provides a method according wherein said vector lacks a 20 sequence encoding ElB 55kD protein capable of binding an E4 34 kDa gene product. In order to produce a gene delivery vehicle according to the invention the vectors according to the invention are propagated in complemanting cells. Thus the invention also 25 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 30 genome of said vector. This is the normal concept for making gene delivery vehicles, except that it now has the advantages as disclosed herein by modification of E2B and/or E4 expression. A gene delivery vehicle according to the invention is defined as any viral particle derived from an 35 adenovirus, a chimaeric adenovirus or comprising adenoviral elements, capable of infecting cells and delivering a gene WO01/20014 PCT/NL00/00640 14 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 s retrovirus, in order to be able to integrate a nucleic acid of interest into a host cell genome. It may even be only a fiber or an adenoviral receptor recognising part of an adenovirus coupled to another virus or non viral vehicle comprising the vector. 10 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. Here the function of the 15 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. As stated herein before, the complementing cell according to 20 the invention preferably also comprises the capability to provide the induction of the E4 and/or E2B products. Thus the invention provides a method wherein said cell further comprises an expression cassette encoding a proteinaceous substance capable of transactivating the inducible 25 (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. To avoid problems associated with the production 30 of replication competent adenoviruses and/or the production of transforming activity in the preparation of gene delivery vehicles it is preferred to apply a method wherein said recombinant vector and said complementing cell have no sequence-overlap that leads to homologous recombination 35 resulting in replication competent adenovirus and/or recombinant adenovirus comprising El sequences.

WO01/20014 PCT/NL00/00640 15 The invention also provides the vectors obtainable by methods as disclosed herein. In one embodiment the invention thus provides a recombinant adenoviral vector lacking ElA and preferably ElB sequences, but having at least the E2B and/or 5 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 io disclosed above. Also provided are 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. Of course such a vector and/or vehicle preferably comprises a therapeutic 15 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 20 disorder or for vaccination purposes. In gene therapy settings one advantage of a recombinant adenoviral vector of the invention 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. 25 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 30 part protected from shut down of expression in the host. A non-limiting example of such a commonly used promoter is the CMV promoter. 35 Detailed description WO01/20014 PCT/NL00/00640 16 The present invention provides means and methods for the generation and manufacturing of recombinant Ad vectors that are modified in E2B and/or E4 functions, preferably, said vectors comprise El and/or E2A deletions. For this purpose, 5 the vector genome is modified in the respective promoter regions, such that the promoter is only active in a suitable complementing cell line or only active following a certain signal in the case of an inducible promoter. The modified promoter is, on the other hand, inactive under normal 10 conditions, and in normal mammalian and/or human cells. Hence, vectors that possess said modified promoter in the E2B and/or E4 region do not express the respective transcription regions in mammalians and/or humans. The promoter modifications can be made in recombinant Ad vector genomes 15 that lack El, or El and E2A, or El and E3, or El and E2A and E3, or El and E4, or El and E2A and E4, or El1 and E3 and E4, or El and E2A and E3 and E4. In one aspect the invention provides the latter four variants in the case of E2B promoter modification only. Since there is no overlap between vector 20 sequences and adenoviral sequences in the complementing cell line, reversion of the modified phenotype due to homologous recombination cannot occur. Successful replacement of the E4 promoter by a synthetic promoter has recently been reported (Fang et al., 1997). In 25 this study, the E4 promoter was replaced by synthetic Gal4 binding sites and the vectors modified in the E4 region could be efficiently propagated in 293 cells expressing the appropriate Gal4/VP16 fusion protein. However, replication of the vectors modified in the E4 region was dramatically 30 impaired in cells that did not express the Gal4/VPl6 fusion protein. It was assumed impossible by persons skilled in the art to manipulate the E2 promoters 'because of overlap with the major late open reading frame L4' (quote-unquote by Rittner 35 et al., 1997). However, in the present invention, we provide means and methods to produce vectors that are attenuated in WO01/20014 PCT/NL00/00640 17 botheE2 early and late promoter regions, in particular in vectors that are also deleted for the E2A gene. For use in gene therapy, it would clearly be s advantageous to have recombinant Ad vectors available that do not produce or produce reduced amounts of viral polypeptides and hence are less immunogenic, but yet retain all capabilities of the current vectors. The present invention provides novel recombinant Ad vectors that improve 10 persistence and diminish pathology in mammals and/or humans. The invention also provides means, methods and materials for the generation, manufacturing and use of such recombinant Ad vectors. In one embodiment of the invention, the genome of an El s15 deleted recombinant Ad vector (as described in W097/00326) is modified in the E4 promoter region. The entire promoter region of the E4 transcription unit is deleted and replaced by a synthetic promoter that consists of an artificial TATA box preceded by a specific sequence that is recognized by the 20 DNA binding domain (DBD) of a DBD-transactivating fusion protein. Said DBD can be derived from a DNA binding protein that originates from prokaryotes and/or lower eukaryotes but not from mammals and/or humans. Alternatively, the DBD can be rationally designed to recognize a synthetic promoter. 25 Transcription of the E4 region of such recombinant Ad genome would not occur in mammalian and/or human cells. As a consequence, such an El deleted /E4 disabled recombinant Ad vector does not produce El encoded polypeptides and produces markedly reduced levels of the E4 encoded polypeptides. In 30 addition, such a vector produces reduced levels of the viral late proteins, since it has been shown that E4 polypeptides are required for the expression of late genes from El deleted vectors (Lusky et al., 1998). In a more preferred embodiment, the genome of an E1 35 deleted recombinant Ad vector is further deleted for E2A and modified in the E4 promoter region. The entire promoter WO01/20014 PCT/NL00/00640 18 region of E4 is deleted from the E1+E2A deleted recombinant Ad DNA and replaced by a synthetic promoter as described above. Transcription of E4 from such a recombinant Ad genome would not occur in mammalian and/or human cells. As a 5 consequence, such an E1+E2A deleted and E4 disabled Ad vector does neither produce El encoded polypeptides, nor the E2A encoded polypeptide and produces markedly reduced levels of the E4 encoded polypeptides. In addition, such a vector expresses reduced levels of viral late genes, since removing 10 E2A and/or disabling E4 functions inhibits the Ad late gene expression from recombinant Ad vectors (see Figure 1A and Lusky et al., 1998). In an even more preferred embodiment, the genome of an E1+E2A deleted recombinant Ad vector is deleted for the E4 15 promoter region and modified in the E2 promoter region. The entire promoter region of the E4 transcription unit is removed from the E1+E2A deleted recombinant Ad DNA and replaced by a synthetic promoter as described above. In addition, the E2 promoter of said vector is inactivated. This 20 inactivation is accomplished by deletion of E2F binding sites from the E2-early promoter, and/or mutation of the TATA box and/or CAP site of the E2-early promoter and/or mutation of the SP1 binding sites and/or TATA-box of the E2-late promoter. A synthetic promoter, as described above, is 25 inserted directly adjacent to the pTP and pol coding sequences of the E2B region. Transcription of the E4 and the E2B region of such recombinant Ad genome would not occur in mammalian and/or human cells. As a consequence, cells infected with said E1+E2A deleted and E2B+E4 attenuated 30 recombinant Ad vector do neither produce El encoded polypeptides, nor the E2A encoded polypeptide, and produce markedly reduced levels of the E2B encoded polypeptides and the E4 encoded polypeptides. In addition, cells infected with these vectors produce reduced levels of viral late gene 35 products since the expression of late genes is abrogated in the absence of E2A and E4 encoded proteins.

WO01/20014 PCT/NL00/00640 19 In another embodiment, the genome of an E1/E2A deleted recombinant Ad vector is modified only in the E2 promoter region, as described above. Transcription of the E2B region of such recombinant Ad genome would not occur in mammalian 5 and/or human cells. Therefore, cells infected with such E1/E2A deleted and E2B attenuated Ad vector do neither produce El-encoded polypeptides, nor the E2A encoded polypeptide, and produce markedly reduced levels of the E2B encoded polypeptides. In addition, cells infected with such o10 vector do not produce viral late encoded proteins since the expression of viral late gene is abrogated in the absence of the E2A encoded protein. In a preferred embodiment, the genome of an E1/E2A deleted and E2B attenuated recombinant Ad vector is further 15 deleted for the entire E4 region, or parts thereof. Cells infected with such E1/E2A/E4 deleted and E2B attenuated Ad vector do neither produce El-encoded polypeptides nor the E2A-encoded polypeptide nor the E4-encoded polypeptides, and produce markedly reduced levels of the E2B-encoded 20 polypeptides. In addition, said infected cells produce reduced levels of viral late gene encoded proteins since expression of viral late genes is abrogated in the absence of E2A- and/or E4-encoded proteins. In another embodiment, all the above-described vectors 25 are deleted for the E3 region of the Ad genome, or parts thereof. A prerequisite for producing Ad vectors that are deleted for the coding sequences of essential genes or that are modified in the promoter region of essential genes is that 30 these defects are complemented in the producing cell lines. Previously, we have established a cell line that efficiently complements the deletion of El (W097/00326). According to the invention, this cell line (designated PER.C6) was further equipped with an expression vector expressing the temperature 35 sensitive mutant of E2A derived from the adenovirus H5ts125, giving rise to PER.C6/E2A. The cell lines allow the WO01/20014 PCT/NL00/00640 20 manufacturing of high titer batches of recombinant Ad vectors deleted for El or double deleted for El and E2A, respectively. Reversion of the El deleted phenotype, i.e. the formation of replication competent Ad (RCA) in PER.C6 or 5 reversion of the El + E2A deleted phenotype in PER.C6/E2A was prevented by elimination of sequence overlap between Ad sequences present in the cell line and sequences present in the recombinant vector. In one embodiment of the invention, the El complementing 10 cell line PER.C6 is equipped with an expression cassette coding for a DBD-transactivation fusion protein. Said DBD recognizes the synthetic sequence preceding the artificial TATA box in the synthetic promoter(s) that are present in the modified genome of the recombinant Ad vectors of the 15 invention. Said DBD is derived from a DNA binding protein that originates from prokaryotes and/or lower eukaryotes but not from mammals and/or humans. Alternatively, the DBD is rationally designed or developed by randomizing techniques to specifically recognize the synthetic promoter. Because said 20 DBD recognizes the synthetic promoter specifically, it does not interfere with other transcription processes in PER.C6 or PER.C6-derived cells and is therefore not toxic. The DBD of the ectopically expressed DNA binding protein is either fused to its native transactivation domain or fused to a 25 transcription activation (TA) domain derived from viral transcription factors (as non-limiting example, the herpes simplex virus VP16 protein) or fused to a TA domain from eukaryotic transcription factors (as non-limiting example, the TA domain of p65-NfkappaB), or fused to a rationally 30 designed TA domain (as non limiting example, an amphipatic a -helix) or fused to a TA domain that is generated by randomizing techniques (as non-limiting example, an acidic blob). Said cell line is suitable for the manufacturing of recombinant Ad vectors that are deleted for El and/or E3 or 35 parts thereof, and in which the E4 promoter region is replaced by a synthetic promoter according to the invention.

WO01/20014 PCT/NL00/00640 21 In a further embodiment, the E1/E2A complementing cell line PER.C6/E2A is equipped with an expression cassette coding for the above-described DBD-transactivating fusion protein. Said cell line is suitable for the manufacturing of 5 recombinant Ad vectors that are deleted for E1+E2A, and that either do or do not have a deletion of the entire E3 region or parts thereof, and in which E4 and/or E2B is equipped with a synthetic promoter according to the invention. In yet another embodiment, the E1+E2A complementing cell 10 line PER.C6/E2A is equipped with an expression cassette coding for the E4-orf6 protein and an expression cassette for the above described DBD-TA fusion protein. Said cell line is suitable for the manufacturing of recombinant Ad vectors that are deleted for El+E2A+E4 or parts thereof and/or E3 or parts 15is thereof, and in which the E2 early and late promoters are knocked-out by mutations and in which E2B is equipped with a synthetic promoter according to the invention. In another aspect of the invention, the E4 and/or E2 promoter sequences of the recombinant Ad vector genome are 20 deleted and appropriately replaced by a so-called inducible promoter, such as, but not limited to, the metallothionein promoter or the mouse mammary tumor virus (MMTV) promoter. Hence, the transcription of the E4 and/or the E2B genes can be switched on or off at will following a specific signal. 25 Said vectors can be produced in complementing cell lines expressing El and/or E2A and/or E4orf-6 in the presence of the signal that triggers the expression of the E4 and/or E2 genes. The vectors according to the invention can be equipped 30 with any foreign nucleic acid, preferably a nucleic acid encoding a therapeutic molecule. The expression can be driven by a strong viral enhancer/promoter such as but not limited to the CMV promoter. More preferably, said expression is driven by strong mammalian promoters such as but not limited 35 to the EF-1 promoter or the UbC promoter to achieve long term expression of the therapeutic gene.

WO01/20014 PCT/NL00/00640 22 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 vitro and in vivo. For instance, the vectors according to the 5 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. For this 10 application, it is of critical importance that the vector itself does not interfere with cellular processes and that it does not overrule the effect of the transgene. The vectors according to the invention are replication defective and do express the remaining viral genes, if at all, only at 15 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. As a non-limiting example, the vectors 20 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. For such application, it is of critical importance that the vector itself does not dominantly elicit a response of the immune system but that 25 the immune response, instead, is directed primarily against the transgene product. Because 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 30 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 35 synthesized and processed in said cells. For this application, it is of importance that the vector itself does WO01/20014 PCT/NL00/00640 23 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 s 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 10 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 15 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. 20 Preferably, 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. In a 25 preferred aspect, 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 30 derived from at least two different adenovirus serotypes. Such vectors are preferred for a variety of reasons based on the observation that in this way at least part of the favourable properties of different adenovirus serotypes may be combined. 35 The invention further provides a method for ex vivo production of a gene product in a cell comprising providing WO01/20014 PCT/NL00/00640 24 said cell with a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to the invention comprising nucleic acid encoding said gene product, culturing said cell to allow expression of said gene s product and optionally harvesting said cell and/or medium said cell was exposed to. In another aspect the invention provides the use of a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to the 10 invention, for the preparation of a medicament. In yet another aspect the invention provides a vaccine comprising a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to the invention, wherein said adenoviral vector comprises a 15 nucleic acid encoding a proteinaceous molecule against which an immune response has to be raised.

WO01/20014 PCT/NL00/00640 25 EXAMPLES The invention will now be described with respect to the generation of recombinant Ad vectors that are deleted for the 5 El and/or E2A and/or E4 region, and that, in addition, harbor E4 and/or E2B genes whose expression is controlled by synthetic promoters as well as with respect to complementing cell lines. However, the scope of the present invention is not intended to be limited thereby. 10 Example 1 Plasmid based system for the generation of recombinant Ad vectors that are deleted for El and/or E2A and in which the 15 E4 and/or E2B promoter is replaced by a synthetic promoter A. Generation of pBr/Ad.Bam-rITR (ECACC deposit P97082122) In order to facilitate blunt end cloning of the ITR 20 sequences, 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 25 preparation was used without further purification in a ligation reaction with pBr322 derived vector DNA prepared as follows: pBr322 DNA was digested with EcoRV and BamHI, de phosphorylated by treatment with TSAP enzyme (Life Technologies) and purified on LMP agarose gel (SeaPlaque 30 GTG). After transformation into competent E.coli DH5a (Life Techn.) and analysis of ampiciline resistant colonies, one clone was selected that showed a digestion pattern as expected for an insert extending from the BamHI site in Ad5 to the right ITR.

WO01/20014 PCT/NL00/00640 26 Sequence analysis of the cloning border at the right ITR revealed that the most 3' G residue of the ITR was missing, the remainder of the ITR was found to be correct. Said missing G residue is complemented by the other ITR during 5 replication. B. Generation of pBr/Ad.Sal-rITR (ECACC deposit P97082119) pBr/Ad.Bam-rITR was digested with BamHI and SalI. The vector 10 fragment including the adenovirus insert was isolated from LMP agarose (SeaPlaque GTG) and ligated to a 4.8 kb Sall BamHI fragment obtained from wt Ad5 DNA and purified with the Geneclean II kit (Bio 101, Inc.). One clone was chosen and the integrity of the Ad5 sequences was determined by 15 restriction enzyme analysis. Clone pBr/Ad.Sal-rITR contains adeno type 5 sequences from the Sali site at bp 16746 up to and including the rITR (missing the most 3' G residue). C. pBr/Ad.Cla-Bam (ECACC deposit P97082117) 20 Wild-type Ad type 5 DNA was digested with Clal and BamHI, and the 20.6-kb fragment was isolated from gel by electro elution. pBr322 was digested with the same enzymes and purified from agarose gel by Geneclean. Both fragments were ligated and transformed into competent DH5a. The resulting 25 clone pBr/Ad.Cla-Bam was analyzed by restriction enzyme digestion and shown to contain an insert with adenovirus sequences from bp 919 to 21566. D. Generation of pBr/Ad.AflII-Bam (ECACC deposit 30 P97082114) Clone pBr/Ad.Cla-Bam was linearized with EcoRI (in pBr322) and partially digested with AflII. After heat inactivation of AflII for 20' at 65 0 C the fragment ends were filled in with WO01/20014 PCT/NL00/00640 27 Klenow enzyme. The DNA was then ligated to a blunt double stranded oligo linker containing a PacI site (5' AATTGTCTTAATTAACCGCTTAA-3'). This linker was made by annealing the following two oligonucleotides: 5' 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 PacI enzyme to remove concatamers of the oligo. The 22016 bp partial fragment containing Ad5 10 sequences from bp 3534 up to 21566 and the vector sequences, was isolated in LMP agarose (SeaPlaque GTG), re-ligated and transformed into competent DH5a. One clone that was found to contain the PacI site and that had retained the large adeno fragment was selected and sequenced at the 5' end to verify 15 correct insertion of the PacI linker in the (lost) AflII site. E. Generation of pBr/Ad.Bam-rlTRpac#2 (ECACC deposit P97082120) and pBr/Ad.Bam-rITR#8 (ECACC deposit P97082121) 20 To allow insertion of a PacI site near the ITR of Ad5 in clone pBr/Ad.Bam-rITR about 190 nucleotides were removed between the ClaI site in the pBr322 backbone and the start of the ITR sequences. This was done as follows: pBr/Ad.Bam-rITR was digested with ClaI and treated with nuclease Bal31 for 25 varying lengths of time (2, 5, 10 and 15 minutes). The extend of nucleotide removal was followed by separate reactions on pBr322 DNA (also digested at the ClaI site), using identical buffers and conditions. Bal31 enzyme was inactivated by incubation at 75 0 C for 10 minutes, the DNA was precipitated 30 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 SalI, satisfactory degradation (-150 bp) was observed in the samples treated for 10 or 15 WO01/20014 PCT/NL00/00640 28 minutes. The pBr/Ad.Bam-rITR samples that were treated for 10 or 15 minutes were then ligated to the above described blunted PacI linkers (See pBr/Ad.AflII-Bam). Ligations were purified by precipitation, digested with excess PacI and 5 separated from the linkers in an LMP agarose gel. After re ligation, DNA's were transformed into competent DH5c and colonies analyzed. Ten clones were selected that showed a deletion of approximately the desired length and these were further analyzed by T-track sequencing (T7 sequencing kit, 10 Pharmacia Biotech). Two clones were found with the PacI linker inserted just downstream of the rITR. After digestion with PacI, clone #2 has 28 bp and clone #8 has 27 bp attached to the ITR. 15 F. Generation of pWE/Ad.Aflll-rITR (ECACC deposit P97082116) Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts. First, a linker containing a unique PacI site was inserted in the EcoRI sites of pWE15 creating pWEl5.pac. To 20 this end, the double stranded PacI oligo as described for pBr/Ad.AflII-BamHI was used but now with its EcoRI protruding ends. The following fragments were then isolated by electro elution from agarose gel: pWEl5.pac digested with PacI, pBr/AflII-Bam digested with PacI and BamHI and pBr/Ad.Bam 25 rITR#2 digested with BamHI and PacI. These fragments were ligated together and packaged using X phage packaging extracts (Stratagene) according to the manufacturer's protocol. After infection of host bacteria, colonies were grown on plates and analyzed for presence of the complete 30 insert. pWE/Ad.AflII-rITR contains all adenovirus type 5 sequences from bp 3534 (AflII site) up to and including the right ITR (missing the most 3' G residue).

WO01/20014 PCT/NL00/00640 29 G. Generation of pWE/Ad.AflII-EcoRI pWEl5.pac was digested with ClaI and 5' protruding ends were filled using Klenow enzyme. The DNA was then digested with PacI and isolated from agarose gel. pWE/AflII-rITR was s digested with EcoRI and after treatment with Klenow enzyme digested with PacI. The large 24-kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the ClaI-digested and blunted pWEl5.pac vector using the Ligation Express t m kit from Clontech. After 10 transformation of Ultra-competent XL10-Gold cells (Stratagene), clones were identified that contained the expected insert. pWE/AflII-EcoRI contains Ad5 sequences from bp 3534-27336. 15 H. Generation of pBR/Ad.AflITI-Bam.tetO-E2B First, 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' 20 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 25 compatible for ligation with HindIII digested DNA. Thus, the product of the annealing reaction was used in a ligation reaction with HindIII digested pGL3-Enhancer Vector (Promega) to yield pAAO-E-TATA. Next, the heptamerized tet-operator sequence was 30 amplified from the plasmid pUHC-13-3 (Gossen and Bujard, 1992) in a PCR reaction using the Expand PCR system (Boehringer) according to the manufacturers protocol. The following primers were used: WO01/20014 PCT/NL00/00640 30 tet3: 5'-CCG GAG CTC CAT GGC CTA ACT CGA GTT TAC CAC TCC C-3' tet5: 5'-CCC AAG CTT AGC TCG ACT TTC ACT TTT CTC-3' The amplified fragment was digested with SstI and HindIII s (SstI and HindIII sites generated in the tet3 and tet5 primers are represented by the nucleotides in bold) and cloned into SstI/HindIII 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 10 plasmid. Next, pAAO-E-TATA-7xtetO was digested with NcoI, and the resulting fragment containing the 7xtetO sequence was purified from agarose and used in a ligation reaction with NcoI digested pNEB.PmAs yielding pNEB.PmAs7xtetO. The plasmid 15 pNEB.PmAs was obtained as follows: pBR/Ad.AflII-Bam (ECACC deposit P97082114) was digested with PmeI/AscI and the resulting PmeI/AscI fragment containing the 5' end of the E2B region was purified from agarose and subcloned into PmeI/AscI digested pNEB 193 (New England BioLabs Inc.) yielding 20 pNEB.PmAs. Then, pNEB.PmAs7xtetO was digested with AscI, Pmel and Scal and the resulting AscI/PmeI 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 PmeI/AscI digested pBR/Ad.AflII-Bam 25 (the fragment that lacks the 5' end of the E2B region) yielding pBR/Ad.AflII-Bam.tetO-E2B. I. Generation of pBR/Ad.Bam-rITRAE2A Deletion of the E2A coding sequences from pBR/Ad.Bam-rITR 30 (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-rITR (ECACC deposit P97082119) in a PCR reaction WO01/20014 PCT/NL00/00640 31 with the Expand PCR system (Boehringer) according to the manufacturers protocol. The following primers were used: Right flanking sequences (corresponding Ad5 nucleotides 24033 to 25180): 5 AE2A.SnaBI: 5'-GGC GTA CGT AGC CCT GTC GAA AG-3' AE2A.DBP-start: 5'-CCA ATG CAT TCG AAG TAC TTC CTT CTC CTA TAG GC-3' The amplified DNA fragment was digested with SnaBI and NsiI (NsiI site is generated in the primer AE2A.DBP-start, 10 underlined). Left flanking sequences (corresponding Ad5 nucleotides 21557 to 22442): AE2A.DBP-stop: 5'-CCA ATG CAT ACG GCG CAG ACG G-3' AE2A.BamHI: 5'-GAG GTG GAT CCC ATG GAC GAG-3' 15 The amplified DNA was digested with BamHI and NsiI (NsiI site is generated in the primer AE2A.DBP-stop, underlined). Subsequently, the digested DNA fragments were ligated into SnaBI/BamHI digested pBr/Ad.Bam-rITR, yielding pBr/Ad.Bam rITRAE2A. The unique NsiI site can be used to introduce an 20 expression cassette for a gene to be transduced by the recombinant vector. J. Generation of pBR/Ad.Bam-rITRAE2AAE2p First, pBR/Ad.Bam-rITRAE2A was digested with AscI and XbaI 25 and the resulting AscI/XbaI fragment containing the E2 promoter region was purified from agarose using the Geneclean II kit and subcloned into AscI/XbaI digested pNEB 193 (New England BioLabs Inc.) yielding pNEB-AX. The latter plasmid contains the Ad E2 early and late promoter sequences. A part 30 of the E2 early promoter sequence was amplified from pNEB-AX using the primers E2eSpeI and E2eSrf whereas a part of the E2 late promoter sequence was amplified from pNEB-AX using the primers E21Aat and E21SpeI.

WO01/20014 PCT/NL00/00640 32 E2eSpeI: 5' GGA CTA GTC TAA GTC TTC TCC AGC GGC CAC ACC CGG 3' E2eSrf: 5' GAG TTA TAC CCT GCC CGG GCG ACC GCA CC 3' 5 E21Aat: 5' GGG CTG TGG ACG TCG GCT TAC CTT CGC AAG TTC GTA CCT GAG GAC TAC CAT GCA CAC GAG ATT AGG 3' E21SpeI: 5' GCG AAA CTA GTC CTT CAG AGT CAG CGC GCA GTA CTT GCT AAA AAG AGC CTC CGC 3' 10 The nucleotides indicated in bold generate the unique Spel, SrfI and AatI restriction sites whereas the underlined nucleotides generate mutations in the promoter sequences that knock out the E2F binding sites and mutate the TATA box and CAP site of the E2 early promoter, and mutate the SP1 binding 15 sites as well as the TATA box of the E2 late promoter. 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 20 extraction, the ligated DNA's were digested with AatII and SrfI. 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 25 then used in a ligation reaction with AatII/SrfI digested pNEB-AX yielding pNEB-AXAE2p. 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. As a 30 result, the modified E2 promoter region that is reconstituted by ligation of the two PCR products is deleted for the above mentioned promoter sequences. Next, pNEB-AXAE2p was digested with AscI and SrfI and the resulting AscI/SrfI fragment containing the mutated E2 promoter was purified from agarose WO01/20014 PCT/NL00/00640 33 using the Geneclean Spin kit and cloned into AscI/SrfI digested pBR/Ad.Bam-rITRAE2A (the fragment that lacks the E2 promoter) giving rise to pBR/Ad.Bam-rITRAE2AAE2p. 5 K. Generation of pBR/Ad.Bam-rITR.tetO-E4, pBR/Ad.Bam-rITR.A E2A.tetO-E4 and pBR/Ad.Bam-rITR.AE2A.AE2p.tetO-E4 First, pBR/Ad.Bam-rITR was digested with PacI and Sse83871 and the resulting PacI/Sse83871 fragment containing the E4 region was purified from agarose using the Geneclean II kit 10 and subcloned into PacI/Sse83871 digested pNEB 193 (New England BioLabs Inc.) giving rise to 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 15 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' 5ITR: ,5' GAT CCG GAG CTC TAC GTC ACC CGC CCC G 3' 20 H3DE4: 5' CCC AAG CTT AGT CCT ATA TAT ACT CGC TC 3' AvDE4: 5' CTC CTG CCT AGG CAA AAT AGC 3' The nucleotides indicated in bold form unique restriction sites for PacI, SstI, HindIII and AvrII, respectively. The PCR products were first purified using the QIAquick PCR 25 purification kit. The PCR product of the sequence downstream of the E4 promoter was digested with HindIII and AvrII. The PCR product of the sequence upstream of the E4 promoter was digested with SstI and ligated to a fragment that contained the 7xtetO sequence which was obtained by digestion of pAAO 30 E-TATA-7xtetO with SstI and HindIII. The ligation products were thereafter digested with HindIII and PacI and the ligation product comprising the region upstream of the E4 promoter linked to the 7xtetO sequence was purified from gel.

WO01/20014 PCT/NL00/00640 34 The latter fragment was used in a ligation reaction with HindIII/AvrII digested PCR product of the sequence downstream of the E4 promoter and with PacI/AvrII digested pNEB-PaSe giving rise to pNEB-PaSe.tetO. 5 Next, pNEB-PaSe.tetO was digested with PacI and Sse83871 and the fragment containing the artificial 7xtetO promoter sequences in front of the E4 region was purified from agarose and cloned into PacI/Sse83871 digested pBR/Ad.Bam-rITRAE2A giving rise to pBR/Ad.Bam-rITRAE2A.tetO-E4. The latter 10 plasmid was used for digestion with PacI and NotI and the resulting NotI/PacI fragment containing the 7xtetO promoter sequences was purified from agarose and used in a ligation reaction with NotI/PacI digested pBR/Ad.Bam-rITR (the fragment that lacks the E4 promoter) giving rise to 15 pBR/Ad.Bam-rITRtetO-E4. Finally, the above described NotI/PacI fragment from pBR/Ad.Bam-rITR.AE2A.tetO-E4 was also cloned into NotI/PacI digested pBR/Ad.Bam-rITR.AE2A.AE2p (the fragment that lacks the E4 promoter region) giving rise to pBR/Ad.Bam-rITR.AE2A.AE2p.tetO-E4. 20 L. Generation of pWE/Ad.AflII-rITR.AE2A.tetO-E2B, pWE/Ad.AflII-rITR.tetO-E4, pWE/Ad.AflII-rITR.AE2A, pWE/Ad.AflII-rITR.AE2A.tetO-E4, and pWE/Ad.AflII-rITR.A E2A.tetO-E2B.tetO-E4. 25 The cosmid pWEl5.pac was linearized by digestion with PacI and used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam.tetO-E2B and BamHI/PacI digested pBR/Ad.Bam rITR.AE2A.AE2p. The ligation mixture was used in a packaging reaction using X phage-packaging extracts (Stratagene) 30 according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII-rITR.AE2A.tetO-E2B.

WO01/20014 PCT/NL00/00640 35 Similarly, PacI-linearized pWEl5.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam and BamHI/PacI digested pBR/Ad.Bam-rITR-tetO-E4. The ligation mixture was used in a packaging reaction using X phage 5 packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII rITR.tetO-E4. Similarly, PacI-linearized pWEl5.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam 10 and BamHI/PacI digested pBR/Ad.Bam-rITR.AE2A. The ligation mixture was used in a packaging reaction using X phage packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII rITR.AE2A. 15 Similarly, PacI-linearized pWEl5.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII-Bam and BamHI/PacI digested pBR/Ad.Bam-rITR.AE2A.tetO-E4. The ligation mixture was used in a packaging reaction using X phage-packaging extracts (Stratagene) according to the 20 manufacturer's protocol, yielding the cosmid pWE/Ad.AflII rITR.AE2A.tetO-E4. Similarly, PacI-linearized pWEl5.pac was used in a ligation reaction with BamHI/PacI digested pBR/Ad.AflII Bam.tetO-E2B and BamHI/PacI digested pBR/Ad.Bam-rITR.AE2A.A 25 E2p.tetO-E4. The ligation mixture was used in a packaging reaction using X phage-packaging extracts (Stratagene) according to the manufacturer's protocol, yielding the cosmid pWE/Ad.AflII-rITR.AE2A.tetO-E2B.tetO-E4. 30 M. Generation of the adapter plasmids 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 WO01/20014 PCT/NL00/00640 36 (introduces a BglII 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 BglII site) and Ad5-2. 5 SV40-1: 5'-GGGGGATCCGAACTTGTTTATTGCAGC-3' SV40-2: 5'-GGGAGATCTAGACATGATAAGATAC-3' Ad5-1: 5'-GGGAGATCTGTACTGAAATGTGTGGGC-3' Ad5-2: 5'-GGAGGCTGCAGTCTCCAACGGCGT-3' 10 Both PCR fragments were digested with BglII and ligated. The ligation product was amplified with primers SV40-1 and Ad5-2 and digested with BamHI and AflII. The digested fragment was then ligated into pMLP.TK predigested with the same enzymes. 15 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. 20 First, a PCR fragment was generated from pZipAMo+PyFl01(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 25 LTR-2: 5'-GCG GAT CCT TCG AAC CAT GGT AAG CTT GGT ACC GCT AGC GTT AAC CGG GCG ACT CAG TCA ATC G-3'. Pwo DNA polymerase (Boehringer Mannheim) was used according to manufacturers protocol with the following temperature cycles: once 5 minutes at 95 0 C; 3 minutes at 55 0 C; and 1 30 minute at 72 0 C, and 30 cycles of 1 minute at 95 0 C, 1 minute at 60 0 C, 1 minute at 72 0 C, followed by once 10 minutes at 720 C. The PCR product was then digested with BamHI and ligated into pMLP10 (Levrero et al., 1991) digested with PvuII and BamHI, thereby generating vector pLTR10. This vector contains 35 adenoviral sequences from bp 1 up to bp 454 followed by a WO01/20014 PCT/NL00/00640 37 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 (PyF101). The promoter fragment was designated L420. Sequencing confirmed correct amplification s of the LTR fragment however the most 5' bases in the pcr fragment were missing so that the PvuII site was not restored. Next, the coding region of the murine HSA gene was inserted. pLTR10 was digested with BstBI followed by Klenow treatment and digestion with NcoI. The HSA gene was obtained 10 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 HSA2, 5'-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3'. 15 The 269 bp-amplified fragment was sub-cloned in a shuttle vector using the NcoI and BglII sites. Sequencing confirmed incorporation of the correct coding sequence of the HSA gene, but with an extra TAG insertion directly following the TAG stop codon. The coding region of the HSA gene, including the 20 TAG duplication was then excised as a NcoI (sticky)-SalI (blunt) fragment and cloned into the 3.5 kb NcoI (sticky)/BstBI (blunt) fragment from pLTR10, resulting in pLTR-HSA10. Finally, pLTR-HSA10 was digested with EcoRI and BamHI after 25 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. This resulted in the new adapter plasmid pAd5/L420-HSA that contains convenient recognition 30 sites for various restriction enzymes around the promoter and gene sequences. SnaBI and AvrII can be combined with HpaI, NheI, KpnI, HindIII to exchange promoter sequences, while the latter sites can be combined with the ClaI or BamHI sites 3' from HSA coding region to replace genes in this construct.

WO01/20014 PCT/NL00/00640 38 The vector pAd5/L420-HSA was then modified to create a SalI or PacI site upstream of the left ITR. Hereto pAd5/L420-HSA was digested with EcoRI and ligated to a PacI linker (5' AATTGTCTTAATTAACCGCTTAA-3'). The ligation mixture was 5 digested with PacI and religated after isolation of the linear DNA from agarose gel to remove concatamerised linkers. This resulted in adapter plasmid pAd5/L420-HSA.pac. Another adapter plasmid that was designed to allow easy exchange of nucleic acid molecules was made by replacing the 10 promoter, gene and polyA sequences in pAd5/L420-HSA with the CMV promoter, a multiple cloning site, an intron and a polyA signal. For this purpose, pAd/L420-HSA was digested with AvrII and BglII followed by treatment with Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vector and 15 adenoviral sequences was isolated and ligated to a blunt 1570 bp fragment from pcDNAl/amp (Invitrogen) obtained by digestion with HhaI and AvrII followed by treatment with T4 DNA polymerase. This adapter plasmid was named pAd5/Clip. To enable removal of vector sequences from the left ITR in 20 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 SalI site and EcoRI overhang. The linker was ligated to the partially digested pAd5/Clip vector and clones were 25 selected that had the linker inserted in the EcoRI site 23 bp upstream of the left adenovirus ITR in pAd5/Clip resulting in pAdS/Clip.sal. To create an adapter plasmid that only contains a polylinker sequence and no promoter or polyA sequences, 30 pAd5/L420-HSA.pac was digested with AvrII and BglII. 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: WO 01/20014 PCT/NL00/00640 39 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'. 5 The annealed linkers were digested with AvrII and BglII and separated from small ends by column purification (Qiaquick nucleotide removal kit) according to manufacturers recommendations. The linker was then ligated to the AvrII/BglII digested pAd5/L420-HSApac fragment. A clone, 10 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 15 that mediates high expression levels in human cells was constructed as follows: pAd5/L420-HSA.pac was digested with AvrII and 5' protruding ends were filled in using Klenow enzyme. A second digestion with HindIII resulted in removal of the L420 promoter sequences. The vector fragment was 20 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 25 CMVminA: 5'-GATCAAGCTTCCAATGCACCGTTCCCGGC-3'. The PCR fragment was first digested with PstI (underlined in CMVplus) after which the 3'-protruding ends were removed by treatment with T4 DNA polymerase. Then the DNA was digested with HindIII (underlined in CMVminA) and ligated into the 30 above described pAd5/L420-HAS.pac vector fragment digested with AvrII and HindIII. The resulting plasmid was named pAd5/CMV-HSApac. This plasmid was then digested with HindIII and BamHI and the vector fragment was isolated and ligated to the polylinker sequence obtained after digestion of pAdMire WO01/20014 PCT/NL00/00640 40 with HindIII and BglII. The resulting plasmid was named pAdApt. Adapter plasmid pAdApt contains nucleotides -735 to +95 of the human CMV promoter (Boshart et al., 1985). The adapter plasmid pCMV.LacZ was generated as 5 follows: The plasmid pCMV.TK (EP 95-202 213) was digested with HindIII, blunted with Klenow and dNTPs and subsequently digested with SalI. The DNA fragment containing the CMV promoter was isolated. The plasmid pMLP.nlsLacZ (EP 95-202 213) was digested with KpnI, blunted with T4 DNA polymerase 10 and subsequently digested with SalI. The DNA fragment containing the LacZ gene and adjacent adenoviral sequences was isolated. Next, the two DNA fragments were ligated with T4 DNA ligase in the presence of ATP, giving rise to pCMV.nlsLacZ. 15 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'. 20 The PCR reaction was performed using Ex Taq (Takara) according to the suppliers protocol at the following amplification program: 5 minutes 94 0 C, 1 cycle; 45 seconds 94 0 C and 30 seconds 60 0 C and 2 minutes 72 0 C, 5 cycles; 45 seconds 94 0 C and 30 seconds 65 0 C and 2 minutes 72°C, 25 25 cycles; 10 minutes 72; 45 seconds 94 0 C and 30 seconds 60'C and 2 minutes 72 0 C, 5 cycles, I cycle. The PCR product was subsequently digested with Kpnl and BamHI and the digested DNA fragment was ligated into KpnI/BamHI digested pcDNA3 (Invitrogen), giving rise to pcDNA3.nlsLacZ. Next, the 30 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.

WO01/20014 PCT/NL00/00640 41 Subsequently, the fragments were ligated, giving rise to pAd/CLIP.LacZ. The reconstitution of the CMV promoter was confirmed by restriction digestion. Next the LacZ gene was digested from pAd/CLIP.LacZ with KpnI and XbaI and isolated 5 from agarose gel. The plasmid pAdApt was digested with KpnI and XbaI and the large fragment was purified from agarose gel. Next, the LacZ fragment and pAdApt fragment were ligated, yielding pAdApt.LacZ. The adapter plasmid pAd5/CLIP.Luc was generated as 10 follows: The plasmid pCMV.Luc (EP 95-202 213) was digested with HindIII and BamHI. The DNA fragment containing the luciferase gene was isolated. The adapter plasmid pAd/CLIP was digested with HindIII and BamHI, and the large fragment was isolated. Next, the isolated DNA fragments were ligated, 15 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 20 following primers: Upstream: 5'-GAT CGA TAT CAC GGC GAG CGC TGC CAC G-3' Downstream: 5'-GAT CGA TAT CTG TCT AAC AAA AAA GCC AAA AAC GGC C-3' (The underlined sequences form EcoRV restriction sites). 25 Next, pAd/CLIP.Sal was digested with Spel to remove the CMV promoter sequences and the remaining part of the vector was religated to yield pAd/LIP.Sal. The latter plasmid was digested with EcoRV and used in a ligation reaction with EcoRV digested Ubiquitin C promoter containing PCR product 30 yielding pAd5/ULIP.Sal. Then, pAd5/ULIP.Sal was digested with NotI, and the resulting overhang was filled in by Klenow treatment and thereafter digested by XbaI, followed by dephosphorylation. The resulting DNA was used in a ligation reaction with KpnI/XbaI digested pAd/CLIP.LacZ.Sal, from WO01/20014 PCT/NL00/00640 42 which the KpnI sites was made blunt by treatment with T4 DNA polymerase in the absence of nucleotides, yielding pAd/ULIP.LacZ.Sal. The adapter plasmid pAd/EF-la.LacZ.Pac is generated as s follows. The EF-la promoter is amplified by PCR from the plasmid pEF-BOS (Mizushima and Nagata, 1990) using the primers Upstream: 5'-GAT CGG TAC CCG TGA GGC TCC GGT GCC C-3' Downstream: 5'-GAT CGG TAC CAA GCT TTT CAC GAC ACC TGA AA 10 TGG-3'. (The underlined sequences form Acc65I restriction sites). Next, pAdApt.LacZ is digested with AvrII and Acc65I, treated with Klenow enzyme and religated to generate pAd5/Nop.LacZ. The latter plasmid is digested with Acc65I and used in a 15 ligation reaction with Acc65I digested PCR product containing the EF-la promoter, yielding pAd5/EF-la.LacZ.

WO01/20014 PCT/NL00/00640 43 Example 2 Generation of producer cell lines for the production of recombinant adenoviral vectors deleted in early region 1 and early region 2A 5 Here is described the generation of cell lines for the production of recombinant adenoviral vectors that are deleted in early region 1 (El) and early region 2A (E2A). The producer cell lines complement for the El and E2A deletion 10 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 15 Protein with has a high affinity for single stranded DNA. Because of its function, constitutive expression of DBP is toxic for cells. The ts125E2A 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 20 fully active at the permissive temperature of 32 0 C, but does not bind to ssDNA at the non-permissive temperature of 39 0 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 0 C. Temperature sensitive E2A 25 gradually becomes functional upon temperature decrease and becomes fully functional at a temperature of 32 0 C, the permissive temperature. A. Generation of plasmids expressing the wild type E2A 30 or temperature sensitive ts125E2A gene. pcDNA3wtE2A: The complete wild-type early region 2A (E2A) coding region was amplified from the plasmid pBR/Ad.Bam-rITR (ECACC deposit P97082122) with the primers DBPpcrl and DBPpcr2 using the Expand T M Long Template PCR 35 system according to the standard protocol of the supplier WO01/20014 PCT/NL00/00640 44 (Boehringer Mannheim). The PCR was performed on a Biometra Trio Thermoblock, using the following amplification program: 94 0 C for 2 minutes, 1 cycle; 94 0 C for 10 seconds + 51 0 C for 30 seconds + 68 0 C for 2 minutes, 1 cycle; 94 0 C for 10 seconds 5 + 58 0 C for 30 seconds + 68 0 C for 2 minutes, 10 cycles; 94 0 C for 10 seconds + 58°C for 30 seconds + 68 0 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 io restriction site (underlined) 5' of the Kozak sequence (italic) and start codon of the E2A coding sequence. The primer DBPpcr2: CGG AAT TCT TAA AAA TCA AAG GGG TTC TGC CGC (5' to 3') contains a unique EcoRI restriction site (underlined) 3' of the stop codon of the E2A coding sequence. 15 The bold characters refer to sequences derived from the E2A coding region. The PCR fragment was digested with BamHI/EcoRI and cloned into BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3wtE2A. pcDNA3tsE2A: The complete ts125E2A-coding region was 20 amplified from DNA isolated from the temperature sensitive adenovirus mutant H5tsl25 (Ensinger et al., 1972; van der Vliet et al., 1975). The PCR amplification procedure was identical to that for the amplification of wtE2A. The PCR fragment was digested with BamHI/EcoRI and cloned into 25 BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3tsE2A. The integrity of the coding sequence of wtE2A and tsE2A was confirmed by sequencing. B. Growth characteristics of producer cells for the 30 production of recombinant adenoviral vectors cultured at 32-, 37- and 390C. PER.C6 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL) and 10mM MgCl 2 in a 10% CO 2 atmosphere WO01/20014 PCT/NL00/00640 45 at either 32 0 C, 37 0 C or 39 0 C. At day 0, a total of 1 x 106 PER.C6 cells were seeded per 25cm 2 tissue culture flask (Nunc) and the cells were cultured at either 32 0 C, 37 0 C or 39 0 C. At day 1-8, cells were counted. Figure 2 shows that the 5 growth rate and the final cell density of the PER.C6 culture at 39 0 C are comparable to that at 370C. The growth rate and final density of the PER.C6 culture at 32 0 C were slightly reduced as compared to that at 370C or 39°C. No significant cell death was observed at any of the incubation 10 temperatures. Thus PER.C6 performs very well both at 32 0 C and 390C, the permissive and non-permissive temperature for ts125E2A, respectively. C. Transfection of PER.C6 with E2A expression vectors; 15 colony formation and generation of cell lines One day prior to transfection, 2 x 106 PER.C6 cells were seeded per 6 cm tissue culture dish (Greiner) in DMEM, supplemented with 10% FBS and 10mM MgCl 2 and incubated at 370C in a 10% CO 2 atmosphere. The next day, the cells were 20 transfected with 3, 5 or 8pg of either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINE

PLUS

Tm Reagent Kit according to the standard protocol of the supplier (Gibco BRL), except that the cells were transfected at 39 0 C in a 10% CO 2 atmosphere. After the transfection, the 25 cells were constantly kept at 39 0 C, the non-permissive temperature for ts125E2A. Three days later, the cells were put in DMEM supplemented with 10% FBS, 10mM MgCl2 and 0.25mg/ml G418 (Gibco BRL), and the first G418 resistant colonies appeared at 10 days post transfection. As shown in 30 table 1, there was a dramatic difference between the total number of colonies obtained after transfection of pcDNA3 (-200 colonies) or pcDNA3tsE2A (~100 colonies) and pcDNA3wtE2A (only 4 colonies). These results indicate that the toxicity of constitutively expressed E2A can be overcome 35 by using a temperature sensitive mutant of E2A (ts125E2A) and WO01/20014 PCT/NL00/00640 46 culturing of the cells at the non-permissive temperature of 390C. From each transfection, a number of colonies was picked by scraping the cells from the dish with a pipette. 5 The detached cells were subsequently put into 24 wells tissue culture dishes (Greiner) and cultured further at 390C in a 10% CO 2 atmosphere in DMEM, supplemented with 10% FBS, 10mM MgCl 2 and 0.25mg/ml G418. As shown in table 1, 100% of the pcDNA3 transfected colonies (4/4) and 82% of the pcDNA3tsE2A 10 transfected colonies (37/45) were established to stable cell lines (the remaining 8 pcDNA3tsE2A transfected colonies grew slowly and were discarded). In contrast, only 1 pcDNA3wtE2A transfected colony could be established. The other 3 died directly after picking. 15 Next, the E2A expression levels in the different cell lines were determined by Western blotting. The cell lines were seeded on 6 well tissue culture dishes and sub-confluent cultures were washed twice with PBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1% 20 SDS in PBS, supplemented with 1mM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein concentrations were determined by the Bio-Rad protein assay, according to 25 standard procedures of the supplier (BioRad). Equal amounts of whole-cell extract were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6 (Reich et al., 1983). The secondary antibody was a 30 horseradish-peroxidase-conjugated goat anti mouse antibody (BioRad). The Western blotting procedure and incubations were performed according to the protocol provided by Millipore. The complexes were visualized with the ECL detection system according to the manufacturer's protocol (Amersham). Figure 3 35 shows that all of the cell lines derived from the pcDNA3tsE2A transfection expressed the 72-kDa E2A protein (left panel, WO01/20014 PCT/NL00/00640 47 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 E2A protein (left panel, lane 2). No E2A protein was detected in extract from a 5 cell line derived from the pcDNA3 transfection (left panel, lane 1), which served as a negative control. Extract from PER.C6 cells transiently transfected with pcDNA3ts125 (left panel, lane 3) served as a positive control for the Western blot procedure. These data confirmed that constitutive 10 expression of wtE2A is toxic for cells and that using the ts125 mutant of E2A could circumvent this toxicity. D. Complementation of E2A deletion in adenoviral vectors on PER.C6 cells constitutively expressing full-length 15 tsl25E2A. The adenovirus Ad5.d1802 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.d1802 was used to test the E2A trans-complementing activity of PER.C6 20 cells constitutively expressing ts125E2A. Parental PER.C6 cells or PER.C6tsE2A clone 3-9 were cultured in DMEM, supplemented with 10% FBS and 10mM MgCl 2 at 39 0 C and 10% CO 2 in 25 cm 2 flasks and either mock infected or infected with Ad5.d1802 at an m.o.i. of 5. Subsequently the infected cells 25 were cultured at 32 0 C and cells were screened for the appearance of a cytopathic effect (CPE) as determined by changes in cell morphology and detachment of the cells from the flask. Full CPE appeared in the Ad5.d1802 infected PER.C6tsE2A clone 3-9 within 2 days. No CPE appeared in the 30 Ad5.d1802 infected PER.C6 cells or the mock infected cells. These data showed that PER.C6 cells constitutively expressing tsl25E2A complemented in trans for the E2A deletion in the Ad5.d1802 vector at the permissive temperature of 32 0

C.

WO 01/20014 PCT/NL00/00640 48 E. Serum-free suspension culture of PER.C6tsE2A cell lines. Large-scale production of recombinant adenoviral vectors for human gene therapy requires an easy and scaleable 5 culturing method for the producer cell line, preferably a suspension culture in medium devoid of any human or animal constituents. To that end, the cell line PER.C6tsE2A c5-9 (designated c5-9) was cultured at 39 0 C and 10% CO 2 in a 175 cm 2 tissue culture flask (Nunc) in DMEM, supplemented with 10 10% FBS and 10mM MgC1 2 . At sub-confluency (70-80% confluent), the cells were washed with PBS (NPBI) and the medium was replaced by 25 ml serum free suspension medium Ex-cellTM 525 (JRH) supplemented with 1 x L-Glutamine (Gibco BRL), hereafter designated SFM. Two days later, cells were detached 15 from the flask by flicking and the cells were centrifuged at 1,000 rpm for 5 minutes. The cell pellet was resuspended in 5 ml SFM and 0.5 ml cell suspension was transferred to a 80 cm 2 tissue culture flask (Nunc), together with 12 ml fresh SFM. After 2 days, cells were harvested (all cells are in 20 suspension) and counted in a Burker cell counter. Next, cells were seeded in a 125 ml tissue culture erlenmeyer (Corning) at a seeding density of 3 x 105 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 0 C in a 10% CO 2 atmosphere. 25 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 106 cells per ml is reached within 5 days of culture. 30 F. Growth characteristics of PER.C6 and PER.C6/E2A at 37 0 C and 39 0 C. PER.C6 cells or PER.C6tsl25E2A (c8-4) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco 35 BRL) supplemented with 10% Fetal Bovine Serum (FBS, Gibco WO01/20014 PCT/NL00/00640 49 BRL) and 10mM MgC1 2 in a 10% CO 2 atmosphere at either 37 0 C (PER.C6) or 39 0 C (PER.C6tsl25E2A c8-4). At day 0, a total of 1 x 106 cells were seeded per 25cm 2 tissue culture flask (Nunc) and the cells were cultured at the respective 5 temperatures. At the indicated time points, cells were counted. The growth of PER.C6 cells at 37 0 C was comparable to the growth of PER.C6ts125E2A c8-4 at 39 0 C (Figure 5). This shows that constitutive expression of ts125E2A encoded DBP had no adverse effect on the growth of cells at the non 10 permissive temperature of 39 0 C. G. Stability of PER.C6ts125E2A For several passages, the PER.C6ts125E2A cell line clone 8-4 was cultured at 390C and 10% CO 2 in a 25 cm 2 tissue 15 culture flask (Nunc) in DMEM, supplemented with 10% FBS and 10 mM MgCl 2 in the absence of selection pressure (G418). At sub-confluency (70-80% confluent), 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 20 1mM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein concentrations were determined by the BioRad protein assay, according to standard procedures of the supplier (BioRad). Equal amounts of whole 25 cell extract were fractionated by SDS-PAGE in 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6 (Reich et al., 1983). The secondary antibody was a horseradish-peroxidase-conjugated goat anti mouse antibody 30 (BioRad). The Western blotting procedure and incubations were performed according to the protocol provided by Millipore. The complexes were visualized with the ECL detection system according to the manufacturer's protocol (Amersham). The expression of ts125E2A encoded DBP was stable for at least 16 35 passages, which is equivalent to approximately 40 cell doublings (Figure 6). No decrease in DBP levels was observed WO01/20014 PCT/NL00/00640 50 during this culture period, indicating that the expression of ts125E2A was stable, even in the absence of G418 selection pressure. 5 Example 3 Generation of tTA expressing packaging cell lines A. Generation of a plasmid from which the tTA gene is expressed. 10 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, pUHD15-1 was digested with EcoRI. The linearized plasmid was treated with Klenow enzyme in the 15 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. Subsequently, the fragment was used in a ligation reaction with BamHI/EcoRV digested pcDNA 3.1 HYGRO (-) (Invitrogen) giving rise to 20 pcDNA3.1-tTA. After transformation into competent E. Coli DH5 a (Life Techn.) and analysis of ampiciline resistant colonies, one clone was selected that showed a digestion pattern as expected for pcDNA3.1-tTA. 25 B. Transfection of PER.C6 and PER.C6/E2A with the tTA expression vector; colony formation and generation of cell lines One day prior to transfection, 2x10 6 PER.C6 or PER.C6/E2A cells were seeded per 60 mm tissue culture dish 30 (Greiner) in Dulbecco's modified essential medium (DMEM, Gibco BRL) supplemented with 10% FBS (JRH) and 10 mM MgCl 2 and incubated at 37 0 C in a 10% CO 2 atmosphere. The next day, cells were transfected with 4-8 pg of pcDNA3.1-tTA plasmid DNA using the LipofectAMINE PLUS T M Reagent Kit according to WO01/20014 PCT/NL00/00640 51 the standard protocol of the supplier (Gibco BRL). The cells were incubated with the LipofectAMINE PLUS

T

m-DNA mixture for four hours at 37 0 C and 10% CO 2 . Then, 2 ml of DMEM supplemented with 20% FBS and 10 mM MgC1 2 was added and cells 5 were further incubated at 37 0 C and 10% CO 2 . The next day, cells were washed with PBS and incubated in fresh DMEM supplemented with 10% FBS, 10 mM MgC1 2 at either 37 0 C (PER.C6) or 39 0 C (Per.C6/E2A) in a 10% CO 2 atmosphere for three days. Then, the media were exchanged for selection 10 media; PER.C6 cells were incubated with DMEM supplemented with 10% FBS, 10 mM MgCl 2 and 50 pg/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 s15 three weeks while nonresistant cells died during this period. From each transfection, 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% 20 FBS, 10 mM MgCl 2 and supplemented with 50 pg/ml (PERC.6 cells) or 100 ig/ml (PERC.6/E2A cells) hygromycin in a 10%

CO

2 atmosphere and at 37 0 C or 39 0 C, respectively. Next, it was determined whether these hygromycin resistant cell colonies expressed functional tTA protein. 25 Therefore, cultures of PER.C6/tTA or PER/E2A/tTA cells were transfected with the plasmid pUHC 13-3 that contains the reporter gene luciferase under the control of the 7xtetO promoter (Gossens and Bujard, 1992). To demonstrate that the expression of luciferase was mediated by tTA, one half of the 30 cultures was maintained in medium without doxycycline. The other half was maintained in medium with 8 gg/ml doxycycline (Sigma). The latter drug is an analogue of tetracycline and binds to tTA and inhibits its activity. All PER.C6/tTA and PER/E2A/tTA cell lines yielded high levels of luciferase, 35 indicating that all cell lines expressed the tTA protein (Figure 7). In addition, the expression of luciferase was WO01/20014 PCT/NL00/00640 52 greatly suppressed when the cells were treated with doxycycline. Collectively, the data showed that the isolated and established hygromycin-resistant PER.C6 and PER/E2A cell clones all expressed functional tTA. 5 Example 4 Generation of recombinant adenoviral vectors. A. El-deleted recombinant adenoviruses with wt E3 sequences 10 To generate El deleted recombinant adenoviruses with the plasmid-based system, the following constructs are prepared: a) An adapter construct containing the expression cassette with the gene of interest linearized with a restriction enzyme that cuts at the 3' side of the overlapping 15 adenoviral genome fragment, preferably not containing any pBr322 vector sequences, and b) A complementing adenoviral genome construct pWE/Ad.AflII rITR (ECACC deposit P97082116) digested with PacI. These two DNA molecules are further purified by 20 phenol/chloroform extraction and ethanol precipitation. Co transfection of these plasmids into an adenovirus packaging cell line, preferably a cell line according to the invention, generates recombinant replication deficient adenoviruses by a one-step homologous recombination between the adapter and the 25 complementing construct. A general protocol as outlined below, and meant as a non-limiting example of the present invention, has been performed to produce several recombinant adenoviruses using various adapter plasmids and the Ad.AflII-rITR fragment. 30 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. Routinely, 40Al lipofectamine, 4Ag adapter plasmid and 4 Ag of the 35 complementing adenovirus genome fragment AflII- rITR (or 2 yg WO01/20014 PCT/NL00/00640 53 of all three plasmids for the double homologous recombination) are used. Under these conditions transient transfection efficiencies of ~50% (48 hrs post transfection) are obtained as determined with control transfections using a 5 pAd/CMV-LacZ adapter. Two days later, cells are passed to ~80 cm 2 flasks and further cultured. Approximately five (for the single homologous recombination) to eleven days (for the double homologous recombination) later a cytopathic effect (CPE) is seen, indicating that functional adenovirus has 10 formed. Cells and medium are harvested upon full CPE and recombinant virus is released from the cells by freeze thawing. An extra amplification step in a 80 cm 2 flask is routinely performed to increase the yield since at the initial stage the titers are found to be variable despite the 15 occurrence of full CPE. After amplification, viruses are harvested and plaque purified using PER.C6 cells. Individual plaques are tested for viruses with active trans-genes. Several different recombinant adenoviruses, comprising the luciferase gene (IG.Ad.CLIP.Luc), the bacterial LacZ gene 20 (IG.Ad.CLIP.LacZ and IG.Ad.CMV.LacZ) or an empty CLIP cassette (IG.Ad.CLIP) have been produced using this protocol. In all cases, functional adenovirus was formed and all isolated plaques contained viruses with the expected expression cassettes. 25 B. Generation of recombinant adenoviruses deleted for early region 1 and early region 2A Besides replacements in the El region, it is possible to delete or replace the E2A region in the adenovirus. This 30 creates the opportunity to use a larger insert or to insert more than one gene without exceeding the maximum packagable size (approximately 105% of wt genome length). Recombinant viruses that are both El and E2A deleted are generated by a homologous recombination procedure as 35 described above for El-replacement vectors using a plasmid based system consisting of: WO01/20014 PCT/NL00/00640 54 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.AflII-rITRAE2A fragment, with or without 5 insertion of a second gene of interest. Generation and propagation of such viruses, e.g. IG.Ad.CMV.LacZAE2A, IG.Ad.CLIP.LacZAE2A, IG.Ad.CLIPAE2A or IG.Ad.CLIP.LucAE2A, requires a complementing cell line for complementation of both El and E2A proteins in trans, as 10 described above. Because 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. 15 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-rITR clone by digestion with XbaI and re 20 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 25 for a heterologous promoter since the transgene is driven by the E3 promoter and pA sequences, leaving more space for coding sequences. To this end, the 2.7-kb EcoRI fragment from wt Ad5 containing the 5' part of the E3 region was cloned into the 30 EcoRI site of pBluescript (KS-) (Stratagene). Next, the HindIII site in the polylinker was removed by digestion with EcoRV and HincII and subsequent re-ligation. The resulting clone pBS.Eco-Eco/ad5AHIII was used to delete the gpl9K coding region. Primers 1 (5'-GGG TAT TAG GCC AA AGG CGC A-3') 35 and 2 (5'-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3') WO01/20014 PCT/NL00/00640 55 were used to amplify a sequence from pBS.Eco-Eco/ad5AHIII corresponding to sequences 28511 to 28734 in wt Ad5 DNA. 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 5 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 NcoI site and subsequently digested with XbaI and MunI. This fragment was then ligated into the pBS.Eco-Eco/ad5AHIII vector that was digested with 10 XbaI (partially) and MunI generating pBS.Eco-Eco/ad5AHIII.A gpl9K. To allow insertion of foreign genes into the HindIII and BamHI site, an XbaI deletion was made in pBS.Eco-Eco/ad5A HIII.Agpl9K to remove the BamHI site in the Bluescript polylinker. The resulting plasmid pBS.Eco-Eco/ad5AHIIIAgpl9K 15 AXbaI, contains unique HindIII and BamHI sites corresponding to sequences 28733 (HindIII) and 29218 (BamHI) in Ad5. After introduction of a foreign gene into these sites, either the deleted XbaI fragment is re-introduced, or the insert is re cloned into pBS.Eco-Eco/ad5AHIII.Agpl9K using HindIII and for 20 example MunI. Using this procedure, we have generated plasmids expressing HSV-TK, hIL-la, rat IL-3, luciferase or LacZ. The unique SrfI and NotI sites in the pBS.Eco-Eco/ad5A HIII. Agpl9K plasmid (with or without inserted gene of interest) are used to transfer the region comprising the gene 25 of interest into the corresponding region of pBr/Ad.Bam-rITR, yielding construct pBr/Ad.Bam-rITRAgp19K (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 30 adenovirus E3 promoter. Recombinant viruses that are both El and E3 deleted are generated by a double homologous recombination procedure for WO01/20014 PCT/NL00/00640 56 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 5 interest, b) the pWE/Ad.AflII-EcoRI fragment, and c) the pBr/Ad.Bam-rITRAgpl9K plasmid with or without insertion of a second gene of interest. In addition to manipulations in the E3 region, changes 10 of (parts of) the E4 region can be accomplished easily in pBr/Ad.Bam-rITR. Moreover, combinations of manipulations in the E3 and/or E2A and/or E4 region can be made. Generation and propagation of such vectors, however, demands packaging cell lines that complement for E2A and/or E4 in trans. 15 C. Generation of El deleted recombinant Ad vectors that possess an attenuated E4 region in PER.C6/tTA cells Recombinant viruses that are El deleted and harbor a synthetic E4 promoter region are generated by a homologous 20 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, 25 b) pWE/Ad.AflII-rITR.tetO-E4 Generation and propagation of such viruses, e.g., IG.Ad/LacZA EltetO-E4 is done in the appropriate complementing cells, i.e. PERC.6/tTA cells. Several different recombinant adenoviruses, comprising the bacterial LacZ gene 30 (IG.Ad.AdApt.LacZ and IG.Ad.ULIP.LacZ) have been produced using this protocol (see table I). D. Generation of recombinant Ad deleted for early region 1 and early region 2A and attenuated for E2B and/or E4 WO01/20014 PCT/NL00/00640 57 Recombinant adenoviral vectors from which both El and E2A are deleted, and which possess E2B and/or E4 regions under transcriptional control of a synthetic promoter are generated by homologous recombination as described above using a 5 combination of the following plasmid DNAs: a) An adapter plasmid for replacement of El, e.g., pULIP LacZ, pAdApt-LacZ, pEF-l-LacZ. b) pWE/Ad.AflII-rITRAE2AtetO-E4; pWE/Ad.AflII-rITRAE2AtetO E2B; pWE/Ad.AflII-rITRAE2AtetO-E2BtetO-E4 10 Generation and propagation of such viruses is done in the appropriate complementing cells, i.e. PER/E2A/tTA cells. Several different recombinant adenoviruses, comprising attenuated E2B or E4 have been produced using this protocol (see table II). 15 E. Growth of 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.AE2AtetO-E4, IG.Ad.ULIP.LacZ.AE2AtetO-E4, 20 IG.Ad.ULIP.LacZ.tetO-E2B, and IG.Ad.ULIP.LacZ.AE2A (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 25 that the growth of IG.Ad.AdApt.LacZ.AE2AtetO-E4, IG.Ad.ULIP.LacZ.AE2AtetO-E4, IG.Ad.ULIP.LacZ.tetO-E2B was drastically impaired in PER/E2A cells whereas these viruses can grow well in PER/E2A/tTA cells. This effect is not due to differences in susceptibility of these cell lines for the 30 virus since the control virus, IG.Ad.ULIP.LacZ.AE2A, did grow very well in PER/E2A cells. Together, this indicated that the E2B and E4 attenuated viruses are strongly disabled in replication in the absence of tTA despite the fact that all other components necessary for replication were available. 35 Together, this result indicates that the attenuation of the WO01/20014 PCT/NL00/00640 58 respective gene regions (E2B and E4) according to the invention has been successful. Example 5 5 Biological activity of IG.Ad/AEltetO-E4, IG.Ad/AE1AE2AtetO E4, IG.Ad/AE1 AE2AtetO-E2B, and IG.Ad/AE1AE2AtetO-E2BtetO-E4 vectors in vitro and in vivo. A. Biological activity of IG.Ad/AEltetO-E4, IG.Ad/AE1AI 10 E2AtetO-E4, IG.Ad/AEl AE2AtetO-E2B, and IG.Ad/AE1AE2AtetO E2BtetO-E4 vectors in vitro In order to demonstrate that El or E1+E2A deleted recombinant Ad vectors with conditionally disabled E2B and/or E4 genes express reduced levels of E2B and/or E4 genes, in 15 mammalian and/or human cells, the following experiment is performed: HeLa cells (ATCC CCL-2) or A549 cells are seeded at 1x10 6 cells per tissue culture plate (Greiner) in DMEM (Gibco BRL) supplemented with 10% FBS (Gibco BRL) in a 10%

CO

2 atmosphere at 37 0 C. The next day, cells are inoculated 20 with 0, 10, 100, 1000 or 10,000 virus particles of IG.Ad/LacZ AEltetO-E4, IG.Ad/LacZAE1AE2AtetO-E4, IG.Ad/LacZAElAE2AtetO E2B or IG.Ad/LacZAElAE2AtetO-E2BtetO-E4 per cell. As a control, parallel cell-cultures are inoculated with 10, 100, 1000, or 10,000 virus particles of IG.Ad/LacZAE1 or 25 IG.Ad/LacZAE1AE2A. Forty-eight hours post inoculation, cells can be either assayed for viral gene (E2, E4 and late genes) expression or for LacZ expression. The LacZ transducing efficiency is determined as follows: Infected cells are washed twice with PBS (NPBI) and 30 fixed for 8 minutes in 0.25% glutaraldehyde (Sigma) in PBS. Subsequently, 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 g] .3H 2 0 (Merck), 5mM

K

3 [Fe(CN) 6 ] (Merck) in PBS. The reaction is stopped by WO01/20014 PCT/NL00/00640 59 removal of the X-gal solution and washing of the cells with PBS. Expression of viral genes is assayed by Western blot analysis using E2B, E4 and viral late protein specific 5 antibodies using the ECL (Amersham) detection system as described by the manufacturer. 10 B. Longevity of transgene-expression from El or E1+E2A deleted recombinant Ad vectors possessing attenuated E2B and/or E4. In order to study whether the replacement of the native E2B and/or E4 promoter with the synthetic promoter increases s15 the longevity of expression of transgene from the recombinant Ad vectors in vivo, the following experiments are performed. A total of 108 or 10' virus particles of either IG.Ad/LacZA El, IG.Ad/LacZAE1AE2A, IG.Ad/LacZAE1tetO-E4, IG.Ad/LacZAE1A E2AtetO-E4, IG.Ad/LacZAE1AE2AtetO-E2B, IG.Ad/LacZAE1A 20 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 25 stained in X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2mM MgCl 2 (Merck), 5mM K4 [Fe(CN) 6 1 .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. It is expected 30 that the expression of LacZ in C57/B16 mice is prolonged when replication-conditioned Ad vectors were used instead of the conventional IG/Ad.LacZAE1 and IG/Ad.LacZAE1AE2A vectors. In contrast, the LacZ expression in the livers of immune deficient NOD-SCID mice is expected to be stable in all 35 cases.

WO01/20014 PCT/NL00/00640 60 Example 6 Residual E4 gene expression from IG.Ad/AE1, IG.Ad/AE1AE2A, IG.Ad/AEltetO-E4, and IG.Ad/AE1AE2AtetO-E4 vectors in vitro 5 In order to demonstrate that El- or El+E2A-deleted recombinant 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 10 were seeded at 1x10 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% CO 2 atmosphere at 37 0 C. The next day, the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLucAE1, IG.Ad/AdAptLucAEAE2A, 15 IG.Ad/AdAptLucAEltetO-E4, or IG.Ad/AdAptLucAElAE2AtetO-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 20 14,000 rpm, 40C 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. For this purpose, equal 25 amounts (30 pg) of total protein from the cleared cell extracts were run in an SDS-polyacrylamide gel and thereafter blotted onto an Immobilon-P membrane (Millipore). This membrane was processed by incubation with a rabbit E4-orf6 specific anti-peptide antiserum (first antibody, 1:2000 30 diluted; Boivin et al., 1999) and a blotting grade affinity purified Goat anti-Rabbit IgG (H+L)-HRP (secondary antibody, 1:7500 diluted; Biorad). The E4-orf6 protein was eventually visualized using the ECL PlusTM Western blotting detection reagents (Amersham Pharmacia Biotech) according to the WO01/20014 PCT/NL00/00640 61 manufacturer's recommendations. The data in Fig.8 and 9 clearly show that the El- and El+E2A-deleted Ad vectors with conditionally disabled E4 genes produced significantly less of the E4-orf6 protein than El- or El+E2A-deleted vectors 5 possessing wt E4. This indicates that the expression of E4, in non-complementing cells, i.e., normal mammalian and/or human cells, is significantly reduced by the replacement of the native E4 promoter by the tet operon. 10 To demonstrate that the reduced expression of E4 was not simply due to a difference in transduction efficiency of the various vectors, a semi-quantitative southern blot analysis of the cell-associated viral genomes was performed. Therefore, total DNA was harvested from the infected cells at 15 30 h post-inoculation by using the Easy-DNA kit (Invitrogen) according to the manufacturer's recommendations. Ten pg of each DNA sample was digested with BamHI, and run in a 0.75% agarose gel. The DNA was thereafter blotted onto a HybondTM N+ nylon transfer membrane (Amersham Pharmacia Biotech) and 20 probed with a HindIII-NheI (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.LucAEltetO-E4, and 25 IG.Ad/AdAptLucAElAE2AtetO-E4 cannot be explained by inefficient transduction. For example, despite the relatively low abundance of viral genomes of the E1+E2A deleted vector, this vector produced significantly more E4-orf6 protein than the vectors possessing conditionally disabled E4. Taken 30 together, these results provide evidence that the attenuation of E4 according to the invention leads to a significant reduction of E4 expression in normal mammalian and/or human cells.

WO01/20014 PCT/NL00/00640 62 Example 7 Residual E2 gene expression from IG.Ad/AE1, IG.Ad/AE1AE2A, IG.Ad/AE1AE2AtetO-E4, and IG.Ad/AE1AE2AtetO-E4 vectors in 5 vitro In order to demonstrate that El- or El+E2A-deleted recombinant Ad vectors with conditionally disabled E4 genes express reduced levels of the E2A (DBP) gene in mammalian 10 and/or human cells, the following experiment was performed: A549 cells were seeded at a density of 1x10 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% CO2 atmosphere at 370C. The next day, the cells were 15 inoculated with 1000 virus particles of IG.Ad/AdAptLucAE1, IG.Ad/AdAptLucAElAE2A, IG.Ad/AdAptLucAElAE2AtetO-E4, or IG.Ad/AdAptLucAElAE2AtetO-E4. At 30 h post-inoculatiop, the cells were harvested as described supra and the cell extracts were analyzed for the presence of the E2A protein DBP by the 20 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; 25 Biorad). The results (Fig.11) clearly show that the El-deleted Ad vector with conditionally disabled E4 produced significantly reduced amounts of the DBP protein in comparison to the El 30 deleted vector possessing wt E4. As expected, the El+E2A deleted vectors produced no DBP protein. Taken together, these results provide evidence that the attenuation of E4 according to the invention leads to a significant reduction WO01/20014 PCT/NL00/00640 63 of E2A expression in normal mammalian and/or human cells. B) In order to demonstrate that El- or El+E2A-deleted recombinant Ad vectors with conditionally disabled E4 genes s express reduced levels of E2B genes in mammalian and/or human cells, the following experiment was performed: A549 cells were seeded at a density of 1x10 6 cells per 10 cm 2 tissue culture dish in DMEM supplemented with 10% FBS, and incubated in a 10% C02 atmosphere at 37 0 C. The next day, the cells were 10 inoculated with 1000 and 10000 virus particles of IG.Ad/AE1, IG.Ad/AElAE2A, or IG.Ad/AElAE2AtetO-E4. 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 15 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 20 genes produced significantly reduced amounts of the pTP protein than El or El+E2A deleted vectors possessing wt E4. Example 8 Residual late gene expression from IG.Ad/AE1, IG.Ad/AE1AE2A, 25 IG.Ad/AE1AE2AtetO-E4, and IG.Ad/AElAE2AtetO-E4 vectors in vitro. In order to demonstrate that El-deleted Ad vectors with conditionally disabled E4 genes express reduced levels of 30 late genes in mammalian and/or human cells, the following experiment was performed: A549 cells were seeded at 1x10 6 cells per 10 cm 2 tissue culture dish in DMEM supplemented with 10% FBS, and incubated in a 10% CO 2 atmosphere at 37 0

C.

WO01/20014 PCT/NL00/00640 64 The next day, the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLucAE1, IG.Ad/AdAptLucAElAE2A, IG.Ad/AdAptLucAElAE2AtetO-E4, or IG.Ad/AdAptLucAElAE2AtetO E4. 5 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. 10 Gerard, Leuven, Belgium) and the blotting grade affinity purified Goat anti-Rabbit IgG (H+L)-HRP (secondary antibody, 1:100000 diluted; Biorad) were used. The results in Fig.13 clearly show that the El- and E1+E2A-deleted Ad vectors with conditionally disabled E4 genes, as well as the vector that 15 was deleted of E1+E2A produced significantly reduced amounts of fiber protein than the vector that was deleted of El only. This shows that attenuation of the expression of the E4 genes by itself causes a reduction in late gene expression, a phenomenon that is also seen in Ad vectors that are deleted 20 of E2A. Example 9 Attenuation of E4 expression leads to a diminished liver toxicity in vivo. 25 To demonstrate that Ad vectors with conditionally disabled E4 are less toxic in vivo than Ad vectors possessing wt E4, mice are injected intravenously via the tail vein with lEll vp of IG.Ad/AE1, IG.Ad/AE1AE2A, IG.Ad/AEltetO-E4, IG.Ad/AElA 30 E2AtetO-E4, or with PBS/0.5% sucrose only. For this purpose, as a non-limiting example, 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 WO01/20014 PCT/NL00/00640 65 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 s 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 10 real-time PCR or Southern blot analysis (to check the transduction efficiency). The remaining 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 15 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. In addition, blood is sampled for blood cell 20 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.

WO01/20014 PCT/NL00/00640 66 Example 10 The effect of the attenuation of E4 on the activity of the CMV promoter driving a transgene is cell-type specific 5 In order to determine the effect of the attenuation of E4 on the activity of the CMV promoter the following experiments have been performed. A549 cells were seeded at 1x10 6 cells per 10 cm 2 tissue culture dish in DMEM supplemented with 10% FBS, and incubated in a 10% CO 2 atmosphere at 37 0 C. The next 10 day, the cells were inoculated with 1000 virus particles of IG.Ad/AdAptLucAE1, IG.Ad/AdAptLucAE1AE2A, IG.Ad/AdAptLucA EltetO-E4, or IG.Ad/AdAptLucAElAE2AtetO-E4. Notably, all these vectors contain the luciferase gene under the control of the CMV promoter. The cells were harvested by detergent 15 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 20 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 E4. This implies that the CMV promoter that drives the 25 expression of the luciferase gene was less active in vectors possessing conditionally disabled E4 than in vectors possessing wt E4. To find out whether the inhibitory effect of the attenuation of E4 on the CMV promoter activity also occurs in other cell 30 types, an experiment, similar to the one described above, was done using primary human endothelial cells. These cells were, for this purpose, inoculated with 1000 vp of IG.Ad/AdAptLucA El or 1000 vp of IG.Ad/AdAptLucAE1AE2AtetO-E4. Forty-eight hours after inoculation, cell extracts were made and examined WO01/20014 PCT/NL00/00640 67 as described above. The results (Fig.15) show that similar amounts of luciferase were found in cells infected with the Ad containing wt E4 and the one possessing conditionally disabled E4. This indicates that the activity of the CMV s promoter is not impaired by the attenuation of E4 in primary human epithelial cells. From these experiments, it is concluded that the effect of the attenuation of E4 on the activity of the CMV promoter is cell type specific. 10 Example 11 The influence of E4 in transgene expression over longer periods of time in lung-, liver and breast tumor derived cell lines 15 To determine whether E4-expression plays a role in transgene expression from the CMV-promoter over longer periods of time in lung and liver derived cell-lines, the following infection- and subsequent luciferase activity experiments 20 were performed. As a part of these experiments, total protein content was also determined. Transduction of A549 (lung derived) cells At day 1, A549 cells were seeded in 96-well plates with a 25 density of 10,000 cells/well in a volume of 100 pl DMEM+10% heat-inactivated FBS and incubated in a humidified CO 2 incubator set at 37oC and 10% CO2. At day 2, the cells were transduced with viruses derived from PER/E2A/tTA cells transfected with the adapter plasmid pAdApt-luc in 30 combination with pWE/Ad.AflII-rITRAE2AtetO-E4 or derived from PER.C6/E2A cells that were transfected with pAdApt-Luc in combination with pWE/Ad.AflII-rITRAE2A. In this transduction experiment crude lysates as well as purified viruses were WO01/20014 PCT/NL00/00640 68 used. For both types of preparations the number of virus particles (vp) 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 5 50,000 vp/cell in a total volume of 150 pl. After transduction, the cells were again incubated in a humidified CO2 incubator set at 370C and 10% CO2. At day 3, cells in plate for time point "24 hour" were washed with 100 pl PBS and lysed with 100 pl lysisbuffer (8 mM MgCl 2 , 1 mM EDTA, 1 10 mM DTT, 1% v/v Triton X-100 and 15% v/v glycerol). Then the plate was frozen at -20'C until luciferase- and total protein assays could be performed. At day 4, cells in plate for time point "48 hour" were washed, lysed and stored according to the protocol described 15 supra. Medium of plates for time points "72 hour", "96 hour" and "168 hour" was removed and new medium was added, to avoid re-infection. At day 5, 6 and 9, cells in plates for respectively time points "72 hour", "96 hour" and "168 hour" were washed, lysed and stored according the protocol 20 described supra. 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 pl sample was put 25 in an appropriate tube after which the machine added 100 pl 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. 30 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 pl was used in the assay. Fig.16 shows the results of the transduction of the A549 WO01/20014 PCT/NL00/00640 69 cells. These data show that the AE2A.tetO-E4 viruses (normal lines) give about a 100 fold lower expression over time as compared to the AE2A viruses (dashed lines). There is no clear decrease in luciferase expression for each virus 5 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. Transduction of HepG2 (liver derived) cells 10 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 pl) and the MOI's 15 that were used were 30 and 300 vp/cell in a total volume of 150 p 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 AE2A.tetO-E4 (normal lines) and 20 the AE2A viruses (dashed lines) are comparable. The luciferase activity derived from both viruses apparently increase over time. Transduction of MCF-7 (breast cancer derived) cells 25 Transduction, 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. 30 Fig.18 shows the luciferase activity results obtained after the transduction of the MCF-7 cells. These results suggest that expression over time of the AE2A.tetO-E4 (normal lines) and the AE2A viruses (dashed lines) are also comparable. The WO01/20014 PCT/NL00/00640 70 result obtained after the AE2A virus infection using a crude lysate with an MOI of 50,000 vp/cell, suggest that there is a drop in expression level after 72 hours. The results obtained after infection with AE2A.tetO-E4 using crude lysates and 5 purified viruses show a slight decrease in luciferase activity after 96 hours.

WO01/20014 PCT/NL00/00640 71 Example 12 Genomic analysis of Ad vectors with conditionally disabled E4 The genomic identity of Ad vectors possessing conditionally 5 disabled E4 was confirmed by Southern blot analysis of the vector genome. For this purpose, the viral genomic DNA was isolated from purified IG.Ad/AE1AE2A and IG.Ad/AElAE2AtetO E4 virus particles. Therefore, 100 l virus suspension containing 1.4x10 11 - 3x10 11 virus particles was mixed with 18 10 p1l buffer (50 mM MgCl 2 , 1.2 mM CaCl 2 , and 130 mM Tris pH 7.5), 9 pl DNaseI (10 mg/ml), and 3 pl H 2 0. This mixture was incubated for 30 min at 37 0 C after which 3.6 p1l EDTA (0.5 M), 4.5 p1l SDS (10%), and 1.5 p1l Proteinase K (20 mg/ml) was added. The mixture was then incubated for 1 h at 50 0 C. The 15 viral DNA was then purified from the mixture using the GeneClean Kit and cut with PstI. Equal amounts of DNA purified from different vectors were run in a 1% agarose gel and blotted onto a HybondTM-N+ nylon transfer membrane and probed with a 313 bp HindIII/NcoI fragment of pNEB-PaSe.tetO. 20 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/AElAE2AtetO-E4 was labeled whereas the genomic DNA of IG.Ad/AElAE2A was unlabeled. This indicates that only 25 the genomic DNA of IG.Ad/AElAE2AtetO-E4 contained the tet operon sequence. The size of the labeled fragment fits the theoretic length of the genomic PstI fragment (2203 bp) containing the rITR and sequences of E4 with the tet operon. From this result, it is concluded that IG.Ad/AElAE2AtetO-E4 30 truly possesses a tet operon in place of the native E4 promoter.

WO01/20014 PCT/NL00/00640 72 Example 13 Serum-free suspension cultures of PER/E2A/tTA and PER.C6/tTA cells. 5 To obtain a serum-free suspension culture of PER/E2A/tTA cells (clone lA1, pn 11), 5x10 6 cells were thawed and cultured in ExCell 525 medium (JRH Bioscienses) supplemented with 4mM L-glutamin with or without the addition of Hygromycin (100 ug/ml). Cells were cultured in T175 flasks. 10 The concentration of Hygromycin is identical to what is used for adherent cultures. However, since the the ExCell 525 medium does not support the culturing of PER.C6 cells and derivatives thereof in the presence of Hygromycin, culturing was continued only with cultures in the absence of the 15 Hygromycin selection pressure. Cultures that were kept in the presence of Hygromycin died after 8 days. Culture conditions in the absence of Hygromycin were as follows: Passage of the cells was done after 2 or 3 days of incubation. A sample of the cells was subsequently taken and 20 counted and stained for determination of the cell density and viability of the cultures. Then, cultures were passed to a new flask or to a roller bottle or diluted in the flask in which the culture was kept. Following this, the culture was sub-cultured to 2x10 5 or to 3x10 5 viable cells per ml and 25 left for another incubation period of 3 or 2 days, depending on the concentration of cells. The cell cultures were further incubated at 37 0 C or at 39 0 C. The standard temperature for incubating adherent cell cultures that contain a temperature sensitive E2A gene (like 30 PER/E2A/tTA) is 39 0 C. The static cultures (in flasks) were incubated at 39 0 C, while the dynamic cultures in roller bottles were incubated at 37 0 C. After 13 population doublings a serum free suspension cell bank of 5x10 6 cells/vial was established. After 11 and 15 WO01/20014 PCT/NL00/00640 73 population doublings, infections using recombinant LacZ expressing and E4-attenuated adenovirus in roller bottles were performed. Roller bottles contained 100 ml suspension cultures with a cell density of approximately 1x10 6 viable 5 cells per ml. These infections yielded functional LacZ expressing virus as was determined after re-infection of the supernatants on A549 cell cultures and subsequent staining of the cells. The dynamic serum free suspension culture was maintained in 10 roller bottles for 61 days, with a total of 44 population doublings, which results in an average population doubling time of 33 hours. The static serum free suspension culture in the flasks was maintained for 34 days, with a total of 19 population doublings, which results in an average population 15 doubling time of 42 hours. Two 2-liter bio-reactor runs were also performed. In the first bio-reactor a run was performed with cells that were kept in the reactor for 8 days with a final density of approximately 4x10 6 cells per ml using standard perfusion 20 conditions known to persons skilled in the art. In the second bio-reactor, cells were cultured for 4 days after which a virus infection was performed while the cell density was approximately 1x10 6 cells per ml. For this an MOI of 70 virus particles per cell was applied with purified 25 attenuated-E4 recombinant adenovirus. After 4 days of infection the bio-reactor run was terminated and the cells and medium were harvested. The experiments to obtain a serum free suspension culture of 30 PER.C6/tTA were similarly executed. For this 5x10 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.

WO01/20014 PCT/NL00/00640 7.4 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 5 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 2x10 5 or 3x10 5 viable cells per ml for an incubation period of 3 or 2 days respectively. All roller bottle (dynamic) cultures were incubated at 370C and flushed 10 with CO2. After 18 population doublings an infection in roller bottles containing 5x10 5 viable cells per ml was performed. The infection was performed with an attenuated-E4 recombinant adenovirus. The obtained virus titer from this infection s15 (taken three days after infection) was 1.2x10 10 virus particles per ml. This number equals 24,000 produced virus particles per cell (seeded cells). After 22 population doublings a serum free suspension cell bank of 5x10 6 cells per vial was established. This was 20 performed after.27 days of culturing resulting in an average population doubling time of 29 hours. Two 2-liter bio-reactor runs were also performed with the PER.C6/tTA cells. In the first bio-reactor cells were cultured for 8 days and reached a density of approximately 25 4x10 6 cells per ml using standard perfusion known to persons skilled in the art. In the second bio-reactor cells reached a density of approximately 3x10 6 cells per ml and were subsequently infected with a concentrated batch of recombinant attenuated-E4 adenovirus that was derived from 30 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 370C. Three days after infection the virus yield in crude samples taken from the bio-reactor was measured. The concentration of this virus WO01/20014 PCT/NL00/00640 75 stock was 7x10 10 virus particles per ml. This equals a production rate of approximately 23,000 virus particles per cell. 5 WO01/20014 PCT/NL00/00640 76 Example 14 Plaque purification, propagation, and titration of Ad vectors comprising conditionally disabled E4 s A. Plaque purification: IG.Ad/AEltetO-E4 and IG.Ad/AElAE2AtetO E4 vectors were plaque-purified by using PER.C6/tTA and PER/E2A/tTA cells, respectively. For this purpose, cells were seeded in 6-wells plates at a density of 1.5x10 6 cells per 10 10 cm 2 well in DMEM + 10% FBS + 10 mM MgCl 2 . After a 4 h incubation at 370C, 10% CO2 (PER.C6/tTA) or 390C, 10% CO2 (PER/E2A/tTA) the medium was replaced by 1 ml of inoculation medium. The inoculation medium consisted of IG.Ad/AEltetO-E4 or IG.Ad/AE1AE2AtetO-E4 vectors that had been diluted in DMEM 15 + 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 370C, 10% CO2 (PER.C6/tTA) or 340C, CO2 (PER/E2A/tTA). Thereafter, the inoculation medium was removed and the cells were washed with PBS and overlaid with 20 3 ml MEM + 10 mM MgCl 2 + 2.5% agarose + 2-5% FBS per well. The cells were further incubated at 370C, 10% CO2 (PER.C6/tTA) or 340C, 10% CO2 (PER/E2A/tTA). Eleven to 14 days later, independent, free plaques were picked using a 20 p1l pipette. Twenty pl of the plaque material was resuspended 25 in 200 pl DMEM + 10% FBS + 10 mM MgC1 2 , and frozen at -200C. B. Propagation of plaque-purified vectors: the above-described resuspended plaque material containing IG.Ad/AEltetO-E4 or 30 IG.Ad/AElAE2AtetO-E4 was thawed and 100 p1l of each plaque material was mixed with 900 pl 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, WO01/20014 PCT/NL00/00640 77 respectively. Cells were incubated at 37 0 C, 10% CO 2 (PER.C6/tTA) or at 34 0 C, 10% CO 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 5 medium. The cell/medium suspension was then frozen at -20 0 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 io 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. C. 15 Determination of vector titers: PER.C6/tTA and PER/E2A/tTA cells were used to determine the infectious titer of batches of IG.Ad/AE1tetO-E4 and IG.Ad/AE1AE2AtetO-E4, respectively. For this purpose, cells were seeded in 96-wells at a density of 4x10 4 cells per well in DMEM + 10% FBS + 10 mM MgCl 2 . The 20 medium was replaced after a 4-hr incubation at 37 0 C, 10% CO 2 (PER.C6/tTA) or 390, 10% CO 2 (PER/E2A/tTA) by 200 pl of serial dilutions (made in DMEM + 10% FBS + 10 mM MgC1 2 ) of IG.Ad/AEltetO-E4 or IG.Ad/AE1AE2AtetO-E4, respectively. Cells were further incubated at 37 0 C, 10% CO 2 and 34oC, 10% 25 CO 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.

WO01/20014 PCT/NL00/00640 78 Example 15 Stability of PER/E2A/tTA cells in producing El+E2A-deleted Ad vectors that are conditionally disabled in E4 5 PER/E2A/tTA (clone 1A1) cells were kept in culture for at least 100 passages in medium containing the selection drug hygromycin (100 pg/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 10 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. For this purpose, 2.5 x 106 cells PER/E2A/tTA cells at passage 17 and at passage 64 were seeded in a 25 cm 2 culture 15 flask (Nunc) in DMEM + 10% FBS + 10 mM MgCl 2 , and incubated at 390C, 10% CO2. The next day, the cells were inoculated with IG.Ad/AdAptLacZAE1AE2AtetO-E4 at an MOI of 200 vp/cell. After a 5-days incubation at 340C and 10% CO2, the progeny vectors were harvested by freeze-thawing the cells and 20 medium. Thereafter, 10-fold dilutions, ranging from 10-1 to 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 370C, the cells were washed twice with PBS and fixed for 8 min with 1% 25 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 K4[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. 30 The percentage of blue cells was thereafter determined. The results (see Table V) revealed that the progeny vectors derived from PER/E2A/tTA at passage 17 and 64 yielded very similar percentages of blue cells at the various dilutions. This indicates that the yields of progeny vector from WO01/20014 PCT/NL00/00640 79 PER/E2A/tTA cells at passage 18 and 65 were very similar. This implies that PER/E2A/tTA cells maintain their capacity to support the propagation of El+E2A-deleted, E4-attenuated vectors over an extensive period of time during which they 5 are kept in culture. Example 16 Construction of pAd/plPspAdapt-eGFP, pAd/plPspAdapt-lacZ, pAd/plPspAdapt-ceNOS, pAd/plPspAdapt-hIL3, pAd/pIPspAdapt 10 hEPO and pAd/plPspAdapt-LacZ pAd5/L420-HSA (described in published PCT patent application WO 99/55132) was digested with AvrII and BglII. The vector fragment was ligated to a linker oligonucleotide digested 15 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 20 TAG GGA TGG C-3'). This ligation resulted in pAdMire. Another batch of pAd5/L420-HSA was also digested with AvrII and 5' protruding ends were filled in using Klenow enzyme. A second digestion with HindIII resulted in removal of the L420 promoter sequences. The vector fragment was isolated and 25 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 30 GCT TCC AAT GCA CCG TTC CCG GC-3'). The PCR fragment was first digested with PstI after which the 3'-protruding ends were removed by treatment with T4 DNA polymerase. Then the DNA was digested with HindIII and ligated into the AvrII/HindIII digested pAd5/L420-HSA vector. The resulting WO01/20014 PCT/NL00/00640 80 plasmid was named pAd5/CMV-HSA. This plasmid was then digested with HindIII and BamHI and the vector fragment was isolated and ligated to the HindIII/BglII polylinker sequence obtained after digestion of pAdMire. The resulting plasmid 5 was named pAdApt. The full length human EPO cDNA (Genbank accession number: M11319) was cloned, employing oligonucleotide primers 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 10 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 15 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 20 BamHI linker sequence. The obtained circular plasmid was checked with KpnI, DdeI and NcoI 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 25 orientation was named pAdApt.EPO and was further digested with HindIII and XbaI restriction enzymes. This EPO insert was isolated and ligated to a HindIII/XbaI digested plPspAdapt6 plasmid (described in WO 99/64582). The resulting plasmid was named pAd/plPspAdapt-hEPO. pIPspAdapt6 plasmids 30 carrying human ceNOS (insert HindIII/XbaI), human IL-3 (insert HindIII/BamHI), LacZ (insert KpnI/BamHI) and eGFP (insert HindIII/EcoRI) were generated via pAd5/CLIP (described in WO 99/55132) and according to methods described in detail in WO 99/64582.

WO01/20014 PCT/NL00/00640 81 Example 17 Miniaturized, multiwell production of E1/E2A deleted and E4 attenuated recombinant adenoviral vectors in PER/E2A/tTA 5 cells Several different PER/E2A/tTA cell clones were tested for the production of recombinant adenoviral viruses with El and E2A deletions in combination with an E4 attenuation. This was 10 performed in 96 well tissue culture plates, to test for usefulness in high-through-put screens in functional genomics. First set of transfections 15 At day 1, eighteen different PER/E2A/tTA cell clones were harvested and diluted in culture medium (DMEM+10% Fetal Bovine Serum and 10 mM MgCl 2 ) to a density of 22,500 cells per 100 l. These suspensions were seeded in two 96-well tissue-culture plates with 100 [il per well in duplo (one 20 clone in four wells divided over two plates). At day 2, one DNA mix was made for each PER/E2A/tTA clone, by diluting 3.9 pg of SalI linearized pAd/Adapt-LacZ and 3.9 pg of PacI linearized pWE/Ad.AflII-rITRAE2A.tetO-E4, in 150 il DMEM. To each DNA mix, 150 pl Lipofectamine mix (38.4 pl Lipofectamine 25 (Life Techn.) + 111.6 pl DMEM) was added. This 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 pl per well) to the PER/E2A/tTA cells, after removal of the medium in which the cells were 30 seeded. After 2 hours incubation in a humidified CO 2 incubator (39 0 C, 10% CO2), 170 pl culture medium was added to each well and the plates were returned to the humidified CO2 incubator (390C, 10% CO2). At day 3, the supernatant in each WO01/20014 PCT/NL00/00640 82 well was replaced with 200 p1l culture medium. The plates were then placed again in another humidified CO2 incubator (340C, 10% CO2). At day 5, one of the duplo 96-wells plates was used to determine the transfection efficiency using LacZ staining 5 (Table III). The LacZ staining procedures are described in PCT/WO99/64582. The other plate was monitored for CPE formation during a period of three weeks after transfection. In Fig.20 the percentage of CPE positive wells, scored three weeks after transfection, is depicted. These results suggest 10 that it was best to continue with PER/E2A/tTA clones 1Al, C101, C103, 2B3, 2C3 and 2D5 for 96 well adenoviral production settings, as outlined supra. These clones were used in a second round transfection procedure. 15 Second set of transfections At day 1, six attached PER/E2A/tTA cell cultures (lAl, 1C1, C103, 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 20 plates for each clone were used to.seed 100 p1 of the cell suspensions per well. At day 2, three different DNA mixes were prepared for each PER/E2A/tTA clone. 1 pg of a linearized adapter molecule (being either pAd/plPspAdapt eGFP, or pAd/plPspAdapt-lacZ or pAd/Adapt-ceNOS) was mixed 25 with 4 pg of PacI linearized pWE/Ad.AflII-rITRAE2A.tetO-E4 in 100 p1 DMEM. To each mix 100 p1 Lipofectamine mix (25.6 p1 Lipofectamine (Life Techn.) + 74.4 p1 DMEM) was added. This DNA/Lipofectamine mixture was left at room temperature for 30 min, after which 1.3 ml DMEM was added. The latter mixtures 30 were then added on top to the PER/E2A/tTA cells in a total volume of 30 p1 per well, after removal of the medium in which the cells were seeded. After 2 hours incubation in a humidified CO2 incubator (390C, 10% CO2), 170 p1 culture WO01/20014 PCT/NL00/00640 83 medium was added to each well and the plates were placed back in the humidified CO2 incubator (390C, 10% CO2). At day 3, the medium of each well was replaced with 200 pl culture medium. The plates were then returned to another humidified CO2 5 incubator (340C, 10% C02). At day 5, one of the two 96-wells plates of each clone that was transfected with the LacZ adapter plasmid was used to determine the transfection efficiency using LacZ staining as described supra. Table IV shows the transfection efficiency of each clone. The other 10 plate for the LacZ transfectants and all other plates were monitored for CPE formation during a period of three weeks after transfection. In Fig.21 the percentage of CPE positive wells, scored three weeks after transfection, is depicted. 15 Example 18 Generation of pBr/Ad.Bam-rITRAE2AtetO-E4.AE3(XbaI) and pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI) To construct E3 deleted versions of the vectors carrying a 20 tetO-E4 attenuation, the following cloning steps were performed. pBr/Ad.Bam-rITRAE2AtetO-E4 was propagated in E. coli strain DM1 (dam-, dcm-) (Life Techn.). The purified plasmid was digested with XbaI, hereby removing the 1.88 kb XbaI-XbaI insert, and subsequently religated. By removing the 25 XbaI-XbaI insert 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 30 resulting plasmid was named pBr/Ad.Bam-rITRAE2AtetO-E4.A E3(XbaI) and was used to construct helper cosmid pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI). pBr/Ad.Bam-rITRA E2AtetO-E4.AE3(XbaI) was digested with BamHI and PacI WO01/20014 PCT/NL00/00640 84 restriction enzymes and an 11 kb fragment, with the XbaI-XbaI deletion, was isolated. The plasmid pWE/Ad.AflII-rITRAE2A was first digested with BamHI and then partially digested with PacI, yielding amongst others a 26.2 kb fragment. The 11 kb 5 BamHI-PacI fragment from and the 26.2 kb fragment were ligated, yielding cosmid pWE/Ad.AflII-rITRAE2AtetO-E4.A E3(XbaI). This cosmid contains sequences identical to that of pWE/Ad.AflII-rITRAE2AtetO-E4 but with the deletion of the XbaI-XbaI fragment. pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI) 10 was used in the production of adenoviruses with El, E2A and E3 deletions and E4 attenuations in 96 well plates.

WO01/20014 PCT/NL00/00640 85 Example 19 Comparison between pWE/Ad.AflII-rITRAE2AtetO-E4 and pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI) in the generation of E4 attenuated viruses in a 96-wells setting for the purpose 5 of functional genomics. At day 1, 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 p 10 1 per well in 96-well-tissue culture plates. At day 2, linearized adapter molecules pAd/plPspAdapt-ceNOS, pAd/plPspAdapt-eGFP, pAd/plPspAdapt-hEPO, pAd/plPspAdapt hIL3, pAd/plPspAdapt-lacZ and pAd/plPspAdapt-luciferase were used for transfection in combination with 2 different PacI 15 linearized helper cosmids: pWE/Ad.AflII-rITRAE2AtetO-E4 and pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI). The DNA transfection procedure was identical to that described in Example (above) describing transfections in a subset of PER/E2A/tTA clones. The transfection efficiency of 20 wells, transfected with lacZ as adapter molecule, scored after lacZ staining, was 70-80% for both cosmid combinations. 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.AflII-rITRA 25 E2AtetO-E4 and pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI) transfections. Subsequently, the wells were subjected to freezing in the culture medium at -200C, followed by thawing and resuspension by repeated pipetting. An aliquot of 100 p1l of the freeze/thawed transfected cells was transferred to 30 each well of a plate with freshly seeded and attached PER/E2A/tTA cells of clone C103. These cells were seeded as described above. The second 96-well plate with PER/E2A/tTA cells, incubated with the freeze/thawed cell lysates of the WO01/20014 PCT/NL00/00640 86 first transfected plate, was checked for CPE formation and stored at -200C. 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 5 with the supernatants from the freeze/thawed transfected cells as shown in Fig.22. The generation of functional virus using this set-up was shown by several assays (described in WO 99/64582) in which the presence of the proteins that are encoded by the adapter plasmids was determined. Fig.24 shows 10 the percentage of functional viruses that produce either human IL-3, LacZ or Luciferase in the 96-wells setting.

WO01/20014 PCT/NL00/00640 87 Example 20 Determination of toxicity using tetO-E4 attenuated viruses in comparison to non-tetO-E4 attenuated viruses using microarray technology 5 To determine the different effects on cellular gene expression and vector associated toxicity between adenoviral vectors with different deletions and attenuations, microarray experiments are performed. This allows the measurement of 10 mRNA expression of thousands of genes simultaneously and the effect a particular adenoviral vectors has on these gene expressions. On day 1, different cell lines, primary as well as 15 established, are seeded in T25 tissue culture flasks. The next day cells are infected with El, E1/E2A, E1/E2A/E3 deleted or E4 attenuated adenoviral vectors with a serie of different MOI's. After 24 hours, medium of the infected cells is changed. Then, 24, 48 and 72 hours after infection cells 20 are harvested and trizol lysates are made from which RNA and subsequently cDNA is generated. This cDNA is used to generate Cy3 and Cy5 labeled probes using common techniques, known to those skilled in the art and described in Zhu et al (1998). Using the wild type El deleted Ad5 vector as a reference, 25 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 30 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 WO 01/20014 PCT/NL00/00640 88 attenuated and non tetO-E4 attenuated vectors.

WO01/20014 PCT/NL00/00640 89 Example 21 In vivo production of nitric oxid upon treatment with E4 attenuated viruses expressing ceNOS. 5 The in vivo duration of NOS3 transgene expression after aerosol gene transfer of rat lungs with the recombinant adenovirus IG.Ad/AE1AE2AtetO-E4-ceNOS was assessed. Twelve male Wistar rats (body weight 300~350 g) were aerosolized via a silastic catheter into the trachea with 10 IG.Ad/AE1AE2AtetO-E4-ceNOS recombinant adenovirus (300 pl physiologic salt solution containing 3x10 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 15 following extubation. At several time points (day 3, 7, 14 and 21) the transduced animals were re-anaesthetized and re-intubated for exhaled Nitric Oxide measurements during room air breathing. Nitric Oxide (NO) levels in exhaled air were measured using standard 20 chemiluminescence (Sievers 280 - NO analyzer NOATM). Each day of exhaled NO measurements, calibration was performed using a calibration-gas-mixture of exactly 400 parts per million (ppm). The minimal detectable NO concentration was 1 ppb. Two animals died 24 hours after the aerosolisation (17%), 25 probably due to traumatic intubation and persistent tracheal damage after extubation. Mean body weight of the transfected animals at baseline was 363±27 g and decreased to 319±15 g (P<0.05 vs baseline) three days after aerosolisation. Seven days after singe aerosol gene transfer, body weight was 30 normalized (325±15 gr, P=NS vs baseline). Three days after aerosolisation, the exhaled NO levels of the aerosolized animals increased from baseline (5±1 ppb) to 14±5 ppb (n=3). Exhaled NO levels remained elevated at day 7 and 14: 12±9 (n=2) and 16±5 ppb (n=4) respectively. After 21 days exhaled WO01/20014 PCT/NL00/00640 90 NO levels returned back to baseline levels, which is approximately 5±1 ppb. These results suggest that there is a longer duration of expression of ceNOS using IG.Ad/AE1AE2AtetO-E4-ceNOS virus as s compared to the duration of ceNOS expression using a first generation (non-attenuated E4) adenoviral ceNOS virus, which was used in the acute and prolonged hypoxia model (Budts et al. 2000). In this model, first generation adenoviral vectors express ceNOS only for one week and the measured 10 concentration of exhaled NO, as determined at day 7 and day 14 returns to baseline levels at day 14. No toxicity of clinical relevance and differences in loss of body weight as compared to non-E4 attenuated vectors, that were used in the past, were detected after gene transfer with the attenuated 15 E4 adenovirus.

WO01/20014 PCT/NL00/00640 91 FIGURE AND TABLE LEGENDS Figure 1: (A) Expression of DBP, Penton and Fiber. A549 cells were infected with a multiplicity of infection 5 (m.o.i.) of either 0, 100, 1,000 or 10,000 vp/cell of IG.Ad/CLIP or IG.Ad.CLIPAE2A. 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 aDBP monoclonal B6, 10 the polyclonal c-Penton base Ad2-Pb571 or the polyclonal a 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, 15 extract from PER.C6 cells infected with IG.Ad.CLIP harvested at starting CPE). In contrast, no DBP, penton-base or fiber was detected in the non-infected A549 cells or cells infected with IG.Ad.CLIPAE2A. These data show that deletion of the E2A gene did not only eliminate residual DBP expression, but also 20 the residual expression of the late adenoviral proteins penton-base and fiber. (B) Residual expression of E4-orf6 and pTP/TP. The blot, as shown in A, was stripped and used to visualize the E4-orf6 and pTP/TP proteins using the method described 25 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. 30 This indicates that the deletion of the E2A gene did not eliminate the residual expression of the E2B and E4 genes. Figure 2: Temperature dependent growth of PER.C6. PER.C6 cells were cultured in Dulbecco's Modified Eagle 35 Medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco WO01/20014 PCT/NL00/00640 92 BRL) and 10mM MgCl 2 in a 10% C0 2 atmosphere at either 32 0 C, 37 0 C or 39 0 C. At day 0, a total of 1 x 106 PER.C6 cells were seeded per 25cm 2 tissue culture flask (Nunc) and the cells were cultured at either 32 0 C, 37 0 C or 39 0 C. At day 1-8, cells 5 were counted. The growth rate and the final cell density of the PER.C6 culture at 390C are comparable to that at 37 0 C. The growth rate and final density of the PER.C6 culture at 32°C were slightly reduced as compared to that at 370C or 39 0 C. 10 PER.C6 cells were seeded at a density of 1 x 10 cells per 25 2 cm tissue culture flask and cultured at either 32-, 37- or 390C. At the indicated time points, cells were counted in a Burker cell counter. PER.C6 grows well at both 32-, 37- and 390C. 15 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 20 Immobilon-P membranes and DBP protein was visualized using the aDBP 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 25 12). In contrast, the only cell line derived from the pcDNAwtE2A transfection did not express the DBP protein (left panel, lane 2). No DBP protein was detected in extract from a cell line derived from the pcDNA3 transfection (left panel, lane 1), which serves as a negative control. Extract from 30 PER.C6 cells transiently transfected with pcDNA3ts125 (left panel, lane 3) served as a positive control for the Western blot procedure. These data confirm that constitutive expression of wtE2A is toxic for cells and that using the ts125 mutant of E2A can circumvent this toxicity.

WO01/20014 PCT/NL00/00640 93 Figure 4: Suspension growth of PER.C6ts125E2A C5-9. The tsE2A expressing cell line PER.C6tsE2A.c5-9 was cultured in suspension in serum free Ex-cell TM. At the indicated time 5 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. Figure 5: Growth curve PER.C6 and PER.C6tsE2A. 10 PER.C6 cells or PER.C6ts125E2A (c8-4) cells were cultured at 37 0 C or 39 0 C, respectively. At day 0, a total of 1 x 106 cells was seeded per 25cm 2 tissue culture flask. At the indicated time points, cells were counted. The growth of PER.C6 cells at 37 0 C is comparable to the growth of PER.C6ts125E2A c8-4 at 15 39 0 C. This shows that constitutive overexpression of ts125E2A has no adverse effect on the growth of cells at the non permissive temperature of 39 0 C. Figure 6: Stability of PER.C6ts125E2A. 20 For several passages, the PER.C6tsl25E2A cell line clone 8-4 was cultured at 39 0 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 25 visualized using the aDBP monoclonal B6 in an ECL detection system. The expression of ts125E2A 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 30 ts125E2A is stable, even in the absence of G418 selection pressure. Figure 7: tTA activity in hygromycin resistent PER.C6/tTA (A) and PER/E2A/tTA (B) cells.

WO01/20014 PCT/NL00/00640 94 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 Ag of pUHC 13-3 (a plasmid that s 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 10 indicated in relative light units (RLU) per Ag protein. Fig.8 Western blot to visualize the residual expression of E4-orf6 (E4-34 kDa) protein and a Southern blot to visualize the 15 cell-associated viral DNA. A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLucAE1 (dEl), IG.Ad/AdAptLucAElA E2A (dE2A), IG.Ad/AdAptLucAEltetO-E4 (dEl.tetO-E4), or IG.Ad/AdAptLucAElAE2AtetO-E4 (dEl.dE2A.tetO-E4). At 30 h post-inoculation, the cells were harvested and the relative 20 amount of E4-orf6 protein in each sample was determined by Western blotting using an E4-orf6 specific anti-peptide serum (kind gift of Dr P. Branton). Parallel cultures, infected by the same vectors at the same time, were used to check the transduction efficiency of the vectors. This was done by 25 Southern analysis. Fig.9 The infections (described for Fig.8) for each analysis were done in triplicate and analyzed by western blotting. 30 Fig.10 The infections (described for Fig.8) for each analysis were done in triplicate and analyzed by Southern blotting.

WO01/20014 PCT/NL00/00640 95 Fig. 11 A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLucAE1 (dEl), IG.Ad/AdAptLucAE1AE2A (dE2A), IG.Ad/AdAptLucAEltetO-E4 (dEl.tetO-E4), or IG.Ad/AdAptLucAE1 5 AE2AtetO-E4 (dEl.dE2A.tetO-E4). All infections were performed in triplicate. At 30 h post-inoculation, 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 10 et al., 1983). In parallel, one culture was analyzed that not been infected with any vector (mock). As a positive control, a lysate of cells in which an Ad vector had undergone replication, had been taken. 15 Fig.12 A549 cells were inoculated with 1000 vp per cell using El deleted Ad vectors (dEl), E1+E2A-deleted Ad vectors (dE2A), El-deleted and E4-attenuated (dEl.tetO-E4), or El+E2A-deleted and E4-attenuated (dEl.dE2A.tetO-E4). At 30 h post 20 inoculation, 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. 25 Fig.13 A549 cells were inoculated with 1000 vp/cell of IG.Ad/AdAptLucAE1 (dEl), IG.Ad/AdAptLucAElAE2A (dE2A), IG.Ad/AdAptLucAEltetO-E4 (dEl.tetO-E4), or IG.Ad/AdAptLucAEl AE2AtetO-E4 (dEl.dE2A.tetO-E4). All infections were performed 30 in triplicate. At 72 h post-inoculation, the cells were harvested and the relative amount of fiber protein in each sample was determined by Western blotting using the polyclonal E641/3 anti-knob domain of fiber (primary antibody, 1:5000 diluted).

WO01/20014 PCT/NL00/00640 96 Fig.14 A549 cells were inoculated with IG.Ad/AdAptLucAEl (dEl), IG.Ad/AdAptLucAE1AE2A (dEl.dE2A), IG.Ad/AdAptLucAEltetO-E4 5 (dEl.tetO-E4), or IG.Ad/AdAptLucAElAE2AtetO-E4 (dEl.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 io cell extracts was measured and expressed in relative light units (RLU) per pg protein present in the cell extracts. Fig.15 Primary human endothelial cells were inoculated with 15 IG.Ad/AdAptLucAE1AE2A (dE2A) or with IG.Ad/AdAptLucAElA 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 20 cell extracts was measured and expressed in relative light units (RLU) per pg protein present in the cell extracts. Fig.16 Longevity of expression in A549 cells of Luciferase upon 25 infection with crude lysates and purified virus in a comparison study with attenuated-E4 and non-attenuated-E4 recombinant adenoviruses. 30 Fig.17 Longevity of expression in HepG2 cells of Luciferase upon infection with crude lysates and purified virus in a comparison study with attenuated-E4 and non-attenuated-E4 WO01/20014 PCT/NL00/00640 97 recombinant adenoviruses. Fig.18 Longevity of expression in MCF-7 cells of Luciferase upon s infection with crude lysates and purified virus in a comparison study with attenuated-E4 and non-attenuated-E4 recombinant adenoviruses. Fig.19 10 Viral genomic DNA was isolated from equal numbers of purified E1+E2A-deleted (Ad.dE2A) and E1+E2A-deleted, E4-attenuated (Ad.dE2A.tetO-E4) Ad vector particles and cut with PstI. As positive controls, the plasmid pWE/Ad.AflII-rITR.tetO-E4 was digested with PacI and PstI, and the plasmid pNEB-PaSe.tetO 15 was digested with HindIII. The DNA samples were run in a 1% agarose gel, blotted onto a membrane and probed with a 32p labeled 313 bp HindIII/NcoI fragment of pNEB-PaSe.tetO. Fig.20 20 Percentage of CPE positive wells, based on the results of the first transfection round. CPE was scored three weeks after transfection. Fig.21 25 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/plPspAdapt eGFP, or pAd/plPspAdapt-lacZ or pAd/Adapt-ceNOS. All three different transfections were in addition to pWE/Ad.AflII-rITR 30 AE2A.tetO-E4. Fig.22 Percentage of cpe positive wells in 96-wells plates using PER/E2A/tTA cells transfected with lacZ adapter plasmid in WO01/20014 PCT/NL00/00640 98 combination with either pWE/Ad.AflII-rITRAE2AtetO-E4 or pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI). Fig.23 5 Percentage of cpe positive wells in 96-wells plates using fresh PER/E2A/tTA cells infected with supernatants from PER/E2A/tTA cells that were transfected with lacZ adapter plasmid in combination with either pWE/Ad.AflII-rITRAE2AtetO E4 or pWE/Ad.AflII-rITRAE2AtetO-E4.AE3(XbaI). 10 Fig.24 Percentage of cpe positive wells harboring viruses that produce functional hIL-3, LacZ and Luciferase. 15 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 20 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 25 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 30 10

-

) 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 0 C. CPE was scored at 4 (A) and 5 (B) days after inoculation. 0 means no CPE; 0-1 means sporadic WO01/20014 PCT/NL00/00640 99 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. 5 CITED LITERATURE Amalfitano A., and J. S. Chamberlain (1997) Isolation and 10 characterization of packaging cell lines that coexpress the adenovirus El, DNA polymerase, and preterminal proteins: implications for gene therapy. Gene Ther. 4;258-263. Armentano, D., J. Zabner, C. Sacks, C. C. Sookdeo, M. P. 15 Smith, J. A. St. George, S. C. Wadsworth, A. E. Smith, and R. J. Gregory (1997) Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J. Virol. 71;2408-2416. 20 Armentano, D., M. P. Smith, C. C. Sookdeo, J. Zabner, M. A. Perricone, J. A. St. George, S. C. Wadsworth, and R. J. Gregory (1999) E4ORF3 requirement for achieving long-term transgene expression from the cytomegalovirus promotor in adenovirus vectors. J. Virol. 73;7031-7034. 25 Blanton, R. A., and T. H. Carter (1979) Autoregulation of adenovirus type 5 early gene expression. III. Transcription studies in isolated nuclei. J. Virol. 29;458-465. 30 Boivin, D., M. R. Morrison, R. C. Marcellus, E. Querido, and P. E. Branton (1999) Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34 kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5. J. Virol. 35 73;1245-1253.

WO 01/20014 PCT/NL00/00640 100 Boshart et al., 1985; A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus, Cell 41,521-530, 1985 5 Bout, A. (1997) Gene therapy. In Pharmaceutical Biotechnology, chapter 7, pp. 167-182. Bridge, E., and G. Ketner (1989) Redundant control of adenovirus late gene expression by early region 4. J. Virol. 10 63;631-638. Brough, D. E., A. Lizonova, C. Hsu, V. A. Kulesa, and I. Kovesdi (1996) A gene transfer vector-cell line system for complete functional complementation of adenovirus early 15 regions El and E4. J. Virol. 70;6497-6501. Brough, D. E., C. Hsu, V. A. Kulesa, G. M. Lee, L. J. Cantolupo, A. Lizonova, and I. Kovesdi (1997) Activation of transgene expression by early region 4 is responsible for a 20 high level of persistent transgene expression from adenovirus vectors in vivo. J. Virol. 71;9206-9213. Budts W., Pokreisz P., Nong Z., Van Pelt N., Gillijns H., Gerard R., Lyons R., Collen D., Bloch K.D. and Janssens S. 25 (2000) Aerosol gene transfer with inducible nitric oxide synthase reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation: in press. Chang, L.-S., and T. Shenk (1990) The adenovirus DNA-binding 30 protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J. Virol. 64;2103-2109. Dedieu, J.-F., E. Vigne, C. Torrent, C. Jullien, I. Mahfouz, 35 J.-M. Caillaud, N. Aubailly, C. Orsini, J.-M. Guillaume, P. Opolon, P. Delaire, M. Perricaudet, and P. Yeh (1997) Long- WO01/20014 PCT/NL00/00640 101 term gene delivery into the livers of immunocompetent mice with El/E4-defective adenoviruses. J. Virol. 71;4626-4637. Dobner, T., N. Horikoshi, S. Rubenwolf, S., and T. Schenk 5 (1996) Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor. Science 272;1470 1473. Ensinger, M. J., and H. S. Ginsberg (1972) Selection and 10 preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J. Virol. 10;328-339. Fang, B., and P. Koch, and J. A. Roth (1997) Diminishing adenovirus gene expression and viral replication by promotor 15 replacement. J. Virol. 71;4798-4803. Filmus, J., J. Remani, and M. H. Klein (1992) Synergistic induction of promoters containing metal- and glucocorticoid responsive elements. Nucleic Acids Res. 20;2755-2760. 20 Fisher, K. J., H. Choi, J. Burda, S.-J. Chen, and J. M. Wilson (1996) Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology 217;11 22. 25 Gossen, M., and H. Bujard (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89;5547-5551. 30 Hardy, S., M. Kitamura, T. Harris-Stansil, Y. Dai, and M. L. Phipps (1997) Construction of adenovirus vectors through cre lox recombination. J. Virol. 71;1842-1849. Huang, M.-M., and P. Hearing (1989) Adenovirus early region 4 35 encodes two gene products with redundant effects in lytic infection. J. Virol. 63;2605-2615.

WO01/20014 PCT/NL00/00640 102 Kaufman, R. J. (1990) Vectors used for expression in mammalian cells. Meth. Enzym. 185;487-511. 5 Kay, R., F. Takei, and R. K. Humphries (1990) Expression cloning of a cDNA encoding Ml/69-Jlld heat-stable antigens. J. Immunol. 145;1952-1959. Kochanek, S., P. R. Clemens, K. Mitani, H.-H. Chen, S. Chan, 10 and C. T. Caskey (1996) A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and 9-galactosidase. Proc. Natl. Acad. Sci. USA 93;5731-5736. 15 Kriegler, M. (1990) Assembly of enhancers, promoters, and splice signals to control expression of transferred genes. Meth. Enzym. 185;512-527. Kumar-Singh R., and J. S. Chamberlain (1996) Encapsidated 20 adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells. Hum. Mol. Genet. 5;913-921. Lanier, L. M., K. Storm, A. Shafaie, and L. E. Volkman (1997) 25 Copper treatment increases recombinant baculovirus production and polyhedrin and pl0 expression. BioTechniques 23;728-735. Leppard, K. N. (1997) E4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections. J. 30 Gen. Virol. 78;2131-2138. Levrero, M., V. Barban, S. Manteca, A. Ballay, C. Balsamo, M. L. Avantaggiati, G. Natoli, H. Schellekens, P. Tiollais, and M. Perricaudet (1991) Defective and nondefective adenovirus 35 vectors for expressing foreign genes in vitro and in vivo. Gene 101;195-202.

WO01/20014 PCT/NL00/00640 103 Lusky, M., M. Christ, K. Rittner, A. Dieterle, D. Dreyer, B. Mourot, H. Schulz, F. Stoeckel, A. Pavirani, and M. Mehtali (1998) In vitro and in vivo biology of recombinant adenovirus 5 vectors with El, E1/E2A, or El/E4 deleted. J. Virol. 72;2022 2032. Marshall A. and Hodgson J. (1998) DNA chips: an array of possibilities. Nature Biotechnology 16:27 10 Mizushima, S., and S. Nagata (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acid Res. 18;5322. Moore, M., N. Horikoshi, and T. Schenk (1996) Oncogenic s15 potential of the adenovirus E4orf6 protein. Proc. Natl. Acad. Sci. USA 93;11295-11301. Nenoi, M., K. Mita, S. Ichimura, I. L. Cartwright, E. Takahashi, M. Yamauchi, and H. Tsuji (1996) Heterogeneous 20 structure of the polyubiquitin gene UbC of HeLa S3 cells. Gene 175;179-185. Nevins, J. R., and J. J. Winkler (1980) Regulation of early adenovirus transcription: a protein product of early region 2 25 specifically represses region 4 transcription. Proc. Natl. Acad. Sci. USA 77;1893-1897. Parks, R. J., and F. L. Graham (1997) A helper-dependent system for adenovirus vector production helps define a lower 30 limit for efficient DNA packaging. J. Virol. 71;3293-3298. Ploegh, H. L. (1998) Viral strategies of immune evasion. Science 280;248-253. 35 Pombo, A., J. Ferreira, E. Bridge, and M. Carmo-Fonseca (1994) Adenovirus replication and transcription sites are WO01/20014 PCT/NL00/00640 104 spatially separated in the nucleus of infected cells. EMBO J. 13;5075-5085. Ramsay G. (1998) DNA chips: State of the art. Nature 5 Biotechnology 16:40 Reich N. C., P. Sarnow, E. Duprey, and A. J. Levine (1983) Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology 10 128;480-484. Rice, S. A., and D. F. Klessig (1985) Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein. J. Virol. 56;767-778. 15 Rittner K., H. Schultz, A. Pavirani, and M. Mehtali (1997) Conditional repression of the E2 transcription unit in El-E3 deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression 20 in vitro. J. Virol. 71;3307-3311. Roth, J., C. K6nig, S. Wienzek, S. Weigel, S. Ristea, and M. Dobbelstein (1998) Inactivation of p53 but not p73 by adenovirus type 5 ElB 55-kilodalton and E4 34-kilodalton 25 oncoproteins. J. Virol. 72;8510-8516. Sandler, A. B., and G. Ketner (1991) The metabolism of host RNAs in cells infected by an adenovirus E4 mutant. Virology 181;319-326. 30 Song, W., H. L. Kong, P. Traktman, and R. G. Crystal (1997) Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors. Hum. Gene Ther. 8;1207-1217. 35 WO01/20014 PCT/NL00/00640 105 Swaminathan, S., and B. Thimmapaya (1995) Regulation of adenovirus E2 transcription unit, p. 177-194. In W. Doerfler and P. B6hm (Eds.), The Molecular Repertoire of Adenoviruses III, Springer-Verlag, Berlin, Germany. 5 Stuart, G. W., P. F. Searle, H. Y. Chen, R. L. Brinster, and R. D. Palmiter (1984) A 12-base-pair DNA motif that is repeated several times in metallotheonein gene promoters confers metal regulation to a heterologous gene. Proc. Natl. 10 Acad. Sci. USA 81;7318-7322. Takekoshi, M., F. Maeda-Takekoshi, S. Ihara, S. Sakuma, and Y. Watanabe (1993) Inducible expression of a foreign gene inserted into the human cytomegalovirus genome. J. Gen. 15 Virol. 74;1649-1652. van der Vliet, P. C. (1995) Adenovirus DNA replication. In W. Doerfler, and P. Bbhm (Eds.) The Molecular Reportoire of Adenoviruses II, Springer Verlag, Berlin, Germany, pp. 1-30. 20 van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginsberg (1975) Thermolabile DNA binding proteins from cells infected with a temperature-sensative mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15;348-354. 25 Weinberg, D. H., and G. Ketner (1986) A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc. Natl. Acad. Sci. USA 80;5383-5386. 30 Yang, Y., Nunes, F. A., K. Berencsi, E. Gonczol, J. F. Engelhardt, and J. M. Wilson (1996) Inactivation of E2A in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7;362-369. 35 WO01/20014 PCT/NL00/00640 106 Yang, Y., Q. Su, and J. M. Wilson (1996) Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J. Virol. 70;7209-7212. 5 Yang, H., and J. Filmus (1996) Insertion of metal-responsive elements increase the inducibility of the mouse mammary tumor virus promoter. Gene 182;177-182. 10 Yarranton, G. T. (1992) Inducible vectors for expression in mammalian cells. Curr. Op. Biotech. 3;506-511. Yeh, P., J.-F. Dedieu, C. Orsini, E. Vigne, P. Denefle, and M. Perricaudet (1996) Efficient dual transcomplementation of 15 adenovirus El and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit. J. Virol. 70;559 565. Zhu H., Cong J-P., Mamtora C., Gingeras T. and Shenk T. 20 (1998) Cellular gene expression altered by human cytomegalovirus: Global monitoring with oligonucleotide arrays. Proc.Natl.Acad.Sci.USA 95:14470 WO 01/20014 PCT/NLOO/00640 107 00 ~ -d i~iz t-4iNt iir too P4i z +- + 47 w N +N+N+ i m,~ WO 01/20014 PCT/NLOO/00640 108 '44 Frii

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WO 01/20014 PCT/NL00/00640 110 Table MIX Transfection efficiency in different PER/E2A/tTA clones, using pAd/Adapt-LacZ in combination with pWE/Ad.AfllI rITRAE2A.tetO-E4 determined upon LacZ staining. pn= passage number. PER/E2A/tTA Blue Clone colored cells lA1 pn 16, 50-60 1Al pn 63 60 1A3 20-50 1A4 40-50 1A6 20-40 1B1 5-20 1B4 5-40 1B5 10-20 1i1i 50-60 1C2 40 1C3 30-40 1C4 10-50 1D4 10 1D6 10-20 2A4 10-20 2A5 20-30 2B3 20-60 2C3 60 2D5 10-20 WO01/20014 PCT/NL00/00640 111 Table IV Transfection efficiency of six different PER/E2A/tTA clones transfected with pAd/plPspAdapt-lacZ and pWE/Ad.AflIl rITRAE2A.tetO-E4 using LacZ staining. pn= passage number PER/E2A/tTA Blue Clone colored cells IA1 pn 16 5 1CI1 5 1C3 50 233 60 2C3 20 2D5 30 WO 01/20014 PCT/NLOO/00640 112 rv v r-4 1 U, LA 00 E-44 N pq- '000o L m Ch 4-) U U4 0 4-) * -r4 -i 4-tn m CU C

Claims

1. 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 5 vector lacking ElA and preferably ElB 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 10 essential products.

2. A method according to claim 1, wherein one such an essential product is open reading frame 1, 3 or 6 of E4.

3. A method according to claim 1 or 2, wherein said vector further comprises an E2B and/or an E4 promoter, wherein said 15 E2B and/or E4 promoter are attenuated through a mutation therein.

4. A method according to claim 1 or 2, wherein E2B and/or E4 is placed under control of at least one, preferably synthetic inducible promotor and/or repressor. 20

5. A method according to any one of claims 1-4, wherein said vector further lacks a functional adenoviral DNA binding protein encoding sequence.

6. A method according to claim 5, wherein said vector lacks a functional E2A region. 25

7. A method according to any one of claims 1-6, wherein said vector lacks a sequence encoding ElB 55kD protein capable of binding an E4 34kDa gene product.

8. A method according to claim 4 wherein said repressor is activated and/or inactivated by an adenoviral DNA binding 30 protein analogue.

9. A method according to any one of claims 1-8, further comprising transducing a complementing cell with said recombinant adenoviral vector wherein said complementing cell WO01/20014 PCT/NL00/00640 114 provides all functions and/or elements essential for replication of said recombinant adenoviral vector, which are lacking in the genome of said vector.

10. A method according to claim 9, wherein said 5 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.

11. A method according to claim 9 or 10, wherein said cell 10 further comprises an expression cassette encoding a proteinaceous substance capable of transactivating the inducible (synthetic) promoter according to claim 4.

12. A method according to claim 11, wherein said proteinaceous substance comprises a DNA binding domain from a 15 prokaryote or a lower eukaryote.

13. A method according to claim 11 or 12 wherein said proteinaceous substance comprises a transactivator domain.

14. A method according to any one of claims 9-13, wherein said recombinant vector and said complementing cell have no 20 sequence-overlap that leads to homologous recombination resulting in replication competent adenovirus and/or recombinant adenovirus comprising El sequences.

15. A recombinant adenoviral vector lacking ElA and preferably EIB sequences, but having at least the E2B and/or 25 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 30 according to any one of claims 1-14.

16. 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, obtainable by a method according to any one of claims 1-14. WO 01/20014 PCT/NL00/00640 115

17. A recombinant adenoviral vector according to claim 15 or a recombinant adenovirus-like gene delivery vehicle according to claim 16, comprising a therapeutic nucleic acid sequence.

18. A recombinant adenoviral vector and/or a recombinant 5 adenovirus-like gene delivery vehicle according to anyone of claims 15-17, wherein said adenoviral vector comprises at least one adeno-associated virus terminal repeat or a functional equivalent thereof.

19. A recombinant adenoviral vector and/or a recombinant 10 adenovirus-like gene delivery vehicle according to anyone of claims 15-18 comprising elements derived from at least two different adenovirus serotypes.

20. A method for ex vivo production of a gene product in a cell comprising providing said cell with a recombinant 15 adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to anyone of claims 15-19 comprising nucleic acid encoding said gene product, culturing said cell to allow expression of said gene product and optionally harvesting said cell and/or medium said cell was 20 exposed to.

21. Use of a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to anyone of claims 15-19, for the preparation of a medicament. 25

22. A vaccine comprising a recombinant adenoviral vector and/or a recombinant adenovirus-like gene delivery vehicle according to anyone of claims 15-19, wherein said adenoviral vector comprises a nucleic acid encoding a proteinaceous molecule against which an immune response has to be raised.