Means and methods for nucleic acid transfer

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MEANS AND METHODS FOR NUCLEIC ACID TRANSFER

Technical Field: The present invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. Specifically, the present invention relates to gene therapy using materials derived from adenovirus, in particular human recombinant adenovirus, and relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses. Furthermore, this invention also pertains to the screening of replication-competent and revertant El and/or E2A adenoviruses from recombinant adenoviruses used in gene therapy.

Background: The current generation of adenoviral vectors for gene therapy contains deletions of the early region 1 (Έ1), where new genetic information can be introduced. The El deletion renders the recombinant virus replication defective. It was generally thought that El-deleted vectors would not express any other adenoviral genes, because El is reported to trigger the transcription of the other adenoviral genes. It has been shown by us and others that these vectors express several early (e.g., E2A) and late genes (e.g., fiber, and penton-base), in the absence of El. This means that delivery of a therapeutic gene using El-deleted adenoviral vectors will result in expression of the therapeutic protein and adenoviral proteins. A cytotoxic immune response is evoked against such transduced cells. It has been shown that cytotoxic T-lymphocytes (“CTLs”) directed against both the transgene product and products encoded by the vector are activated, following vector administration into immunocompetent animals (Song et al., Hum. Gene Ther. 8: 1207, 1997; Yang et al., J. Virol. 70: 7209, 1996). Activated CTLs subsequently eradicate transduced cells from the recipient. Consistent with this, the longevity of transgene expression is significantly extended in immuno-deficient and immuno-compromised animals.

Expression of at least some adenoviral genes in a target cell is at least in part due to background replication of the recombinant adenoviral vector genome and/or background activity of promoters driving the respective adenoviral genes (Yang et al., Nature Genet. 7: 362, 1994; Lusky et al.. J.

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Virol. 72: 2022, 1998). As a result of the expression of at least some adenovirus proteins in a target cell in a recipient, an immune response may be mounted against transduced cells. Such an immune response is often not desired, especially when long-term expression of a transgene is aimed for. One mechanism by which adenovirus proteins in a target cell in a recipient may cause the immune system of the recipient to remove the target cell is the following. Proteins encoded by expressed adenovirus genes can be processed into small peptides in a proteasome of the target cell. Peptides produced during this processing can subsequently be presented at the cell surface of the transduced cells in the complex of MHC class-I and bb2-microglobulin molecules. Finally, one or more of the peptides may be recognized as non-self peptides by circulating CTLs whereupon transduced cells can be eradicated from the recipient (reviewed in Ploegh, Science 280: 248, 1998).

Description of the Invention

In one aspect the present invention provides at least in part a solution to the problem of undesired removal of target cells in a recipient.

The present invention also provides, at least in part, a solution for the immune response against viral proteins. To this end, the invention provides improved recombinant adenoviral vectors that, in addition to deletion of El, are also deleted for the adenoviral early 2A gene (“E2A gene” or Έ2Α”). The protein encoded by E2A is expressed from recombinant El-deleted adenoviral vectors. In addition to that, residual expression of E2A from El-deleted recombinant adenoviral vectors induces the expression of the viral late genes, since DNA binding protein (“DBP”) has a positive regulatory effect on the adenovirus major late promoter (“MLP”) and, therefore, on the expression of the late genes (Chang et al., J. Virol. 64: 2103, 1990). Deletion of the E2A gene from the recombinant adenoviral genome will therefore improve the characteristics of recombinant adenoviral vectors. First, deletion of E2A will eliminate the synthesis of DBP. Second, it will inhibit the background replication of the recombinant adenoviral backbone. Third, it will reduce the residual expression of the late genes. Finally, it will increase the capacity of the vector to harbor larger and/or multiple transgenes.

The E2A gene encodes the 72-kDa protein single stranded DBP whose activity is pivotal for the adenovirus DNA replication (reviewed in The Molecular Repertoire of Adenoviruses II, Springer-Verlag 1995). Therefore,

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PCT/NL00/00263 manufacturing of vectors that are deleted for E2A requires a cell line that complements for the deletion of E2A in the recombinant adenoviral vector. Major hurdles in this approach are:

I. that E2A should be expressed to very high levels and

II. that constitutive expression of E2A is toxic for cells and, therefore, impossible to achieve (Klessig et ah, Mol. Cell Biol. 4: 1354, 1984).

The current invention, therefore, involves the use of a temperature sensitive mutant of E2A derived from a temperature sensitive adenovirus under control of strong viral enhancer sequences, e.g., the cytomegalovirus enhancer for the generation of E2A complementing cell lines. DBP (tsl25E2A) from hAd5tsl25 is inactive at 39θϋ, but is fully active at 32θΟ. High levels of this protein can be maintained in the new complementing cells of the invention at the non-permissive temperature, until the switch is made to the permissive temperature. The invention also provides means and methods to use the complementing cell line, comprising E2A, tsE2A, or both El and tsE2A, for the generation of E2A- or El- and E2A-deleted adenoviral vectors. The invention also involves inducible expression of E2A or tsE2A.

The invention also provides new cell lines that complement for E2A or for both the El and the E2A deletion in the vector. The invention also provides new recombinant adenoviral vectors deleted for E2A or both El and E2A.

Brief Description of the Figures

FIG. 1 depicts the temperature dependent growth of PER.C6. PER.C6 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL) and lOmM MgCb in a 10% CO2 atmosphere at either 32°C, 37°C or 39°C. At day 0, a total of 1 x 10^G PER.C6 cells were seeded per 25cm² tissue culture flask (Nunc) and the cells were cultured at either 32°C, 37°C or 39°C. At day 1-8, cells were counted. The growth rate and the final cell density of the PER.C6 culture at 39°C are comparable to that at 37°C. The growth rate and final density of the PER.C6 culture at 32°C were slightly reduced as compared to that at 37°C or 39°C.

FIG. 2 depicts 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

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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 15 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 PER.C6 cells transiently transfected with pcDNA3ts!25 (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 tsl25 mutant of E2A can circumvent this toxicity.

FIG. 3 depicts DBP expression in pcDNA3tsl25E2A transfected 293 cells. Equal amounts of whole-cell extract were fractionated by SDS-PAGE on

10% gels. Proteins were transferred onto Immobilon-P membranes and DBP protein was visualized using the aDBP monoclonal B6 in an ECL detection system. Clone 20 (lane 8) from the pcDNA3tsl25E2A transfected 293 cells expressed the full-length tsl25E2A encoded 72-kDa DBP. No E2A encoded DBP was detected in extract from a cell line (clone 4) derived from the pcDNA3 transfected 293 cells (lane 1), which serves as a negative control. Extract from PER.C6 cells stably expressing tsl25E2A encoded DBP (polyclonal cell line 5) (lane 2) served as a positive control for the Western blot procedure. The other 293 clones either did not express tsl25E2A encoded DBP (clones 21 and 22, lanes 9 and 10 respectively) or expressed aberrant products running with a faster (clones 3, 12, 16 and 18 lanes 4-7) or slower (clone 2, lane 3) mobility in SDS/PAGE.

FIG. 4 depicts suspension growth of PER.C6tsl25E2A cell line c5-9. PER.C6tsl25E2Ac5-9 cells were seeded in a 125ml tissue culture Erlenmeyer at a seeding density of 3 x 10⁵ cells per ml in a total volume of 20 ml serum free medium. Cells were further cultured at 125 RPM on an orbital shaker at 39°C in a 10% CO2 atmosphere. Cells were counted at day 1-6. The mean growth curve from 8 cultures is shown. PER.C6tsl25E2Ac5-9 performs 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.FIG. 5 depicts growth curve PER.C6 and PER.C6tsE2A. PER.C6 cells or PER.C6tsl25E2A (c8-4) cells were cultured at 37°C or 39°C, respectively. At day 0, a total of 1 x 10⁶

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PCT/NL00/00263 cells was seeded per 25cm² tissue culture flask. At the indicated time points, cells were counted. The growth of PER.C6 cells at 37°C is comparable to the growth of PER.C6tsl25E2A c8-4 at 39°C. This shows that constitutive overexpression of tsl25E2A has no adverse effect on the growth of cells at the non-permissive temperature of 39°C.

FIG. 6 depicts stability of PER.C6tsl25E2A. For several passages, the PER.C6tsl25E2A cell line clone 8-4 was cultured at 39°C in medium without G418. Equal amounts of whole-cell extract from different passage numbers were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes and DBP protein was visualized using the aDBP monoclonal B6 in an ECL detection system. The expression of tsl25E2A encoded DBP is stable for at least 16 passages, which is equivalent to approximately 40 cell doublings. No decrease in DBP levels were observed during this culture period, indicating that the expression of tsl25E2A is stable, even in the absence of G418 selection pressure.

FIG. 7 depicts revertant-free manufacturing of DE1/E2A vectors. The recombinant adenoviral vector DNA was screened for reversion of the E2A deleted phenotype by PCR. As shown in the left panel, E2A sequences were amplified from the DNA samples (+) and control samples (-) spiked with both 1, 10 and 40 molecules using primer set A, as evidenced by the amplification of a 260 base pair (“bp”) DNA fragment. In contrast, no E2A sequences were amplified from the non-spiked samples, showing that reversion of the E2Adeleted did not occur. As shown in the right panel, the PCR reactions with primer set B yielded the expected DNA fragment of 169 bp in the samples containing the recombinant adenoviral vector DNA (+). From the negative control samples containing the water instead of DNA (-), no DNA fragment of 169 bp was amplified. These data show, that elimination of overlap between adenoviral sequences in the vector and cell line prevents reversion of the E2Adeleted phenotype.

FIG. 8 depicts transduction of HeLa cells with IG.Ad/CMV.LacZ and IG.Ad/CMV.LacZDE2A. HeLa cells were infected with a multiplicity of infection (“m.o.i.”) of either 0, 10, 100 or 1000 viral particles IG.Ad/CMV.LacZ or IG.Ad/CMV.LacZDE2A per cell. Forty-eight hours post infection, cells were stained with X-gal solution . IG.Ad/CMV.LacZDE2A transduced HeLa cells stained at least as good as did IG.Ad/CMV.LacZ, at all m.o.i.’s.

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FIG. 9 depicts luciferase activity in infected A549 and HeLa cells. HeLa and A549 cells were infected with a m.o.i. of either 0, 10, 100, 1,000 or 10,000 virus particles (“vp”) IG.Ad/CLIP.Luc or IG.Ad/CLIP.LucDE2A per cell. Two days post infection, cells were lysed and the luciferase activity was determined. Both the IG.Ad/CLIP.LucDE2A infected HeLa and A549 cells produce more luciferase enzyme than the IG.Ad/CLIP.Luc infected HeLa and A549 and HeLa cells, at all m.o.i.’s tested.

FIG. 10 depicts the expression of DBP, Penton and Fiber. A549 cells were infected with a m.o.i. of either 0, 100, 1,000 or 10,000 vp/cell

IG.Ad/CLIP or IG.Ad.CLIPDE2A. Seventy-two hours post infection, cell extracts were prepared and equal amounts of whole cell extract were fractionated by SDS-PAGE on 10% gels. The proteins were visualized with the aDBP monoclonal B6, the polyclonal a-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, 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.CLIPDE2A.

These data show that deletion of the E2A gene did not only eliminate residual DBP expression, but also the residual expression of the late adenoviral proteins penton-base and fiber.

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Best Mode of the Invention

According to a presently preferred embodiment of the invention, a cell according to the invention is capable of at least, in part, complementing adenovirus E2A function of an adenovirus defective in E2A function. Such a cell includes a nucleic acid encoding adenovirus E2A or a functional part, derivative and/or analogue thereof, integrated into the genome of the cell. Preferably, the cell has E2A nucleic acid derived from a temperature sensitive adenovirus such as but not limited to adenovirus tsl25. More preferably, such a cell further includes a nucleic acid encoding adenovirus El-region proteins or a functional part, derivative and/or analogue thereof. Such a cell could be derived from the “PER.C6 cell line (commercially available from IntroGene, bv, and deposited, under ECACC deposit accession number 96022940 under the provisions of the Budapest Treaty with the Centre for Applied Microbiology and Research Authority (European Collection of Animal Cell

Cultures), Porton Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an International Depository Authority, in accordance with the Budapest Treaty, on_, 199_).

The invention also includes a method for producing an adenovirus particle containing an adenovirus vector with a functional deletion of E2A.

Such a method involves providing a cell as previously described with the functionally deleted adenovirus vector, culturing the cell, and harvesting the virus particle. In such a method, the functional deletion can comprise a deletion of at least part of the nucleic acid encoding E2A. In such a method, the nucleic acid encoding adenovirus E2A in the genome of the cell, the promoter or polyA sequences preferably have no sequence overlap with the vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising E2A function. In the method, the adenovirus vector preferably further comprises a functional deletion of Elregion encoding nucleic acid, comprising providing one of the previously described cells with the adenovirus vector, culturing the cell and harvesting the virus particle. In such a method, the nucleic acid encoding adenovirus Elregion preferably does not comprise sequence overlap with the vector which leads to replication competent adenovirus and/or to the formation of an adenovirus vector comprising an El function. Furthermore, in the method, the adenovirus vector further comprises at least one nucleic acid of interest.

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The invention also includes an adenovirus vector comprising a functional deletion of adenovirus E2A. Such a functional deletion is preferably a deletion of at least part the nucleic acid encoding E2A. The deletion may encompass the entire coding region of E2A. Such an adenovirus vector preferably includes a deletion corresponding to a deletion of nucleotides 22443 to 24032 in adenovirus 5. The deletion can include a deletion of nucleic acid encoding El-region proteins. The deletion of nucleic acid encoding El-region proteins can comprise a deletion corresponding to a deletion of nucleotides 459 to 3510 in adenovirus 5. Again, the adenovirus vector preferably further includes at least one nucleic acid of interest.

An adenovirus vector according to the invention can, but does not necessarily, also comprise at least a deletion of a region which in adenovirus 5 corresponds to nucleotides 22418-24037 or a deletion of a region which in adenovirus 5 corresponds to nucleotides 22443-24032. Such vectors can further comprise at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22347 and/or nucleotides 24060 until the right ITR or at least 3534-22417 and/or 24038 until the right ITR or at least nucleic acid which in adenovirus 5 corresponds to nucleotides 3534-22442 and/or nucleotides 24033 until the right ITR.

The invention also includes preparations of adenovirus vector containing adenovirus particles wherein the adenovirus vector comprises a functional deletion of E2A. Such an adenovirus vector preferably further includes a deletion of nucleic acid encoding El-region proteins, and may be free of adenovirus vectors comprising E2A function. In such a case the preparation may be free of adenovirus vectors comprising nucleic acid encoding a functional E2A, or a functional part, derivative and/or analogue thereof. The preparation is preferably free of adenovirus vectors comprising nucleic acid encoding El-region proteins or parts, derivatives and/or analogues thereof.

The invention also includes a method for providing cells of an individual with a nucleic acid of interest, without risk of administering simultaneously a replication competent adenovirus vector, comprising administering the individual one of the previously described preparations.

The invention is further described by the use of the following illustrative

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Examples.

Example I

Generation of producer cell lines for the production of recombinant adenoviral vectors deleted in El and E2A or El and E2A

Here is described the generation of cell lines for the production of recombinant adenoviral vectors that are deleted in El and E2A. The producer cell lines complement for the El and E2A deletion from recombinant adenoviral vectors in trans by constitutive expression of the El and E2A genes, respectively. The pre-established Ad5-El transformed human embryo retinoblast cell line PER.C6 (commercially available from IntroGene, bv of Leiden, NL, see also International Patent Appln. WO 97/00326) and Ad5 transformed human embryo kidney cell line 293 (Graham et ah, J. Gen. Virol. 36: 59, 1977) were further equipped with E2A expression cassettes.

The adenoviral E2A gene encodes a 72 kDa DBP with has a high affinity for single stranded DNA. Because of its function, constitutive expression of DBP is toxic for cells. The tsl25E2A mutant encodes a DBP which has a Pro -> Ser substitution of amino acid 413 (van der Vliet, J. Virol. 15: 348, 1975). Due to this mutation, the tsl25E2A encoded DBP is fully active at the permissive temperature of 32°C, but does not bind to ssDNA at the non-permissive temperature of 39°C. This allows the generation of cell lines that constitutively express E2A, which is not functional and is not toxic at the non-permissive temperature of 39°C. Temperature sensitive E2A gradually becomes functional upon temperature decrease and becomes fully functional at a temperature of 32°C, the permissive temperature.

A. Generation of plasmids expressing the wild type E2A- or temperature sensitive tsl25E2A gene.

pcDNA3wtE2A: The complete wild-type E2A coding region was amplified from the plasmid pBR/Ad.Bam-rITR (ECACC deposit P97082122) with the primers DBPpcrl and DBPpcr2 using the Expand™ Long Template PCR system according to the standard protocol of the supplier (Boehringer Mannheim). The PCR was performed on a Biometra TRIO THERMOBLOCK, using the following amplification program: 94°C for 2 minutes, 1 cycle; 94°C for 10 seconds + 51 C for 30 seconds + 68°C for 2 minutes, 1 cycle; 94° C for 10 seconds + 58 C for 30 seconds + 68°C for 2 minutes, 10 cycles; 94°C for 10

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PCT/NL00/00263 seconds + 58°C for 30 seconds + 68°C for 2 minutes with 10 seconds extension per cycle, 20 cycles; 68°C for 5 minutes, 1 cycle. The primer DBPpcrl: CGG GAT CCG CCA CCA TGG CCA GTC GGG AAG AGG AG (5’ to 3’) (SEQ ID NO:1) contains a unique BamHI restriction site (underlined) 5’ of the Kozak sequence (itahc) 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’) (SEQ ID NO:2) contains a unique EcoRI restriction site (underlined) 3’ of the stop codon of the E2A coding sequence. The bold characters refer to sequences derived from the E2A coding region. The PCR fragment was digested with

BamHI/EcoRI and cloned into BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3wtE2A.

pcDNA3tsE2A: The complete tsl25E2A-coding region was amplified from DNA isolated from the temperature sensitive adenovirus mutant H5tsl25 (Ensinger et al., J. Virol. 10: 328, 1972; van der Vliet et al., J. Virol.

15: 348, 1975). The PCR amplification procedure was identical to that for the amplification of wtE2A. The PCR fragment was digested with BamHI/EcoRI and cloned into BamHI/EcoRI digested pcDNA3 (Invitrogen), giving rise to pcDNA3tsE2A. The integrity of the coding sequence of wtE2A and tsE2A was confirmed by sequencing.

B. Growth characteristics of producer cells for the production of recombinant adenoviral vectors cultured at 32-, 37- and 39°C.

PER.C6 cells were cultured in Dulbecco’s Modified Eagle Medium 25 (“DMEM”, Gibco BRL) supplemented with 10% FBS and lOmM MgCh in a

10% CO2 atmosphere at either 32°C, 37°C or 39°C. At day 0, a total of 1 x 10^GPER.C6 cells were seeded per 25cm² tissue culture flask (Nunc) and the cells were cultured at either 32°C, 37°C or 39°C. At day 1-8, cells were counted.

FIG. 1 shows that the growth rate and the final cell density of the PER.C6 culture at 39°C are comparable to that at 37°C. The growth rate and final density of the PER.C6 culture at 32°C were slightly reduced as compared to that at 37°C or 39°C. No significant cell death was observed at any of the incubation temperatures. Thus PER.C6 performs very well both at 32°C and 39°C, the permissive and non-permissive temperature for tsl25E2A, respectively.

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C. Transfection of PER.C6 and 293 with E2A expression vectors; 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 lOmM MgCb and incubated at 37°C in a 10% CO2 atmosphere. The next day, the cells were transfected with 3, 5 or 8gg of either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA per dish, using the LipofectAMINE PLUS™ Reagent Kit according to the standard protocol of the supplier (Gibco BRL), except that the cells were transfected at 39°C in a 10% CO2 atmosphere. After the transfection, the cells were constantly kept at 39°C, the non-permissive temperature for tsl25E2A. Three days later, the cells were put on DMEM, supplemented with 10% FBS, lOmM MgCb and 0.25mg/ml G418 (Gibco BRL) and the first G418 resistant colonies appeared at 10 days post transfection. As shown in 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 constitutive expression of E2A is toxic and the toxicity of constitutively expressed E2A can be overcome by using a temperature sensitive mutant of E2A (tsl25E2A) and culturing of the cells at the nonpermissive temperature of 39°C.

Table 1: Number of colonies after transfection of PER.C6 with E2A expression vectors:

<td> plasmid</td><td> number of colonies</td><td> cell lines established</td>

<td> pcDNA3</td><td> ~ 200</td><td> 4/4</td>

<td> PcDNA3wtE2</td><td> 4</td><td> 1/4</td>

<td> A</td><td></td><td></td>

<td> PcDNA3tsE2A</td><td> -100</td><td> 37/45</td>

PER.C6 cells were transfected with either pcDNA3, pcDNA3wtE2A or pcDNA3wtE2A and cultured in selection medium containing 0.25 mg/ml G418 at 39°C. All colonies (4/4) picked from the pcDNA3 transfection and 82% (37/45) of the colonies from the pcDNA3tsE2A transfection were established to stable cell lines. In contrast, only 25% (1/4) of the colonies from the pcDNA3wtE2A transfection could be established to a cell line.

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From each transfection, a number of colonies was picked by scraping the cells from the dish with a pipette. The detached cells were subsequently put into 24 wells tissue culture dishes (Greiner) and cultured further at 39°C in a 10% CO2 atmosphere in DMEM, supplemented with 10% FBS, lOmM MgCh and 0.25mg/ml G418. As shown in Table 1, 100% of the pcDNA3 transfected colonies (4/4) and 82% of the pcDNA3tsE2A 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 pcDNA3wtE2Atransfected colony could be established. The other 3 died directly after picking.

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% SDS in PBS, supplemented with ImM phenylmethylsulfonylfluoride and

0.1 mg/ml trypsin inhibitor). After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein concentrations were determined by the Bio-Rad protein assay, according to standard procedures of the supplier (BioRad). Equal amounts of whole-cell extract were fractionated by SDS20 PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6 (Reich et al., Virology 128: 480, 1983). The secondary antibody was a horseradishperoxidase-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). FIG. 2 shows that all of the cell lines derived from the pcDNA3tsE2A transfection express the 72-kDa E2A protein (left panel, lanes 4-14; middle panel, lanes 1-13; right panel, lanes 1-12). In contrast, the only cell line derived from the pcDNAwtE2A transfection did not express the E2A protein (left panel, lane 2). No E2A 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 PER.C6 cells transiently transfected with pcDNA3tsl25 (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 tsl25 mutant of E2A can circumvent this toxicity.

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In contrast to PER.C6 cells, the culturing of 293 cells at 39°C is troublesome. Therefore, the transfection of 293 cells with either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A was performed at 37°C in an atmosphere of 10% CO2, a semi-permissive temperature for tsl25E2A encoded DBP. A day prior to transfection, 293 cells were seeded in DMEM, supplemented with 10% FBS and lOmM MgCb, at a density of 3.6 x 10⁵ cells per 6 cm tissue culture dish (Greiner). Five hours before transfection, cells received fresh medium. Cells were transfected with 7.2gg of either pcDNA3, pcDNA3wtE2A or pcDNA3tsE2A plasmid DNA using the Calcium Phosphate Transfection

System according to the standard protocol of the supplier (Gibco BRL). Two days post transfection, cells were put on selection medium, i.e., DMEM supplemented with 10% FBS, lOmM MgCh and 0.1 mg/ml G418. The first colonies appeared at day 12 post transfection. As shown in Table 2, total number of colonies obtained after transfection of pcDNA3 (-100 colonies) or pcDNA3tsE2A (-25 colonies) was significantly higher than that obtained after transfection of pcDNA3wtE2A (only 2 colonies). A total of 22 clones from the pcDNA3tsE2A transfection were picked by scraping the cells from the dish with a pipette. The detached cells were subsequently put into 96 wells tissue culture dishes (Greiner) and cultured further at 37°C in a 10% CO2 atmosphere in DMEM, supplemented with 10% FBS, lOmM MgCb and

O.lmg/ml G418. 16 out of the 22 picked colonies could be established as cell lines (the 6 remaining colonies grew badly and were discarded).

Table 2: Number of colonies after transfection of 293 with E2A expression vectors:

<td> plasmid</td><td> number of colonies</td>

<td> pcDNA3</td><td> ~ 100</td>

<td> PcDNA3wtE2A</td><td> 2</td>

<td> PcDNA3tsE2A</td><td> 25</td>

Selection of colonies derived from 293 cells transfected with E2A expression cassettes. Cell line 293 was transfected with either pcDNA3, pcDNA3wtE2A or pcDNA3wtE2A and cultured in selection medium containing 0.1 mg/ml

G418at37°C.

Next, the E2A expression level in 8 different cell lines was determined by Western blotting. The cell lines were seeded on 6 well tissue culture dishes

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WO 01/07571 PCT/NLO0/OO263 and sub-confluent cultures were washed twice with PBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS, supplemented with ImM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein concentrations were determined by the BioRad protein assay, according to standard procedures of the supplier (BioRad). Equal amounts of whole-cell extract were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6 (Reich et al., Virology 128:

480, 1983). The secondary antibody was a 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). FIG. 3 shows, that, in contrast to the

PER.C6tsE2A cell lines, only clone 20 (lane 8) from the pcDNA3tsE2A transfected 293 cells expressed the full-length tsl25E2A encoded 72-kDa DBP. No E2A encoded DBP was detected in extract from a cell line (clone 4) derived from the pcDNA3 transfected 293 cells (lane 1), which serves as a negative control. Extract from PER.C6 cells stably expressing tsl25E2A encoded DBP (polyclonal cell line 5) (lane 2) served as a positive control for the Western blot procedure. The other 293 clones either did not express tsl25E2A encoded DBP (clones 21 and 22, lanes 9 and 10 respectively) or expressed aberrant products running with a faster (clones 3, 12, 16 and 18 lanes 4-7) or slower (clone 2, lane 3) mobility in SDS/PAGE. These results show that generation of E2A complementing cell line by using temperature sensitive mutants of E2A is not specific for PER.C6 cells, but that it applies to eukaryotic cells in general (e.g., 293 cells). In addition, the 293 data show that keeping the temperature sensitive E2A encoded DBP as inactive as possible is crucial for easy generation of such cell lines. The 293 cell lines were generated at an intermediate temperature of 37°C, a temperature at which tsl25E2A encoded DBP is only partially inactivated. This explains the high number of cell lines expressing aberrant DBP products.

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D. Complementation of E2A deletion in adenoviral vectors on PER.C6- and 293 cells constitutively expressing full-length tsl25E2A encoded DBP.

The adenovirus Ad5.dl802 is an Ad 5 derived vector deleted for the major part of the E2A coding region and does not produce functional DBP (Rice et al., J. Virol. 56: 767, 1985). Ad5.dl802 was used to test the E2A transcomplementing activity of PER.C6 cells constitutively expressing tsl25E2A. Parental PER.C6 cells or PER.C6tsE2A clone 3-9 were cultured in DMEM, supplemented with 10% FBS and lOmM MgCb at 39°C and 10% CO2 in 25 cm²flasks and either mock infected or infected with Ad5.dl802 at an m.o.i. of 5. Subsequently the infected cells were cultured at 32°C and cells were screened for the appearance of a cytopathic effect (CPE) as determined by changes in cell morphology and detachment of the cells from the flask. Table 3 shows that full CPE appeared in the Ad5.dl802 infected PER.C6tsE2A clone 3-9 within 2 days. No CPE appeared in the Ad5.dl802 infected PER.C6 cells or the mock infected cells. These data show that PER.C6 cells constitutively expressing tsl25E2A complement in trans for the E2A deletion in the Ad5.dl802 vector at the permissive temperature of 32°C.

These cells are therefore suitable for production of recombinant adenoviral vector that are deficient for functional E2A.

Table 3: Complementation of E2A deletion in adenoviral vectors on

PER.C6 cells and PER.C6 cells constitutively expressing temperature sensitive E2A.

<td></td><td> 32°C day 2</td>

<td> PER.C6 mock</td><td> -</td>

<td> PER.C6 dl802</td><td> -</td>

<td> PER.C6tsl25c3-9 mock</td><td> -</td>

<td> PER.C6tsl25c3-9 dl802</td><td> Full CPE</td>

Parental PER.C6 cells or PER.C6tsl25E2A clone 3-9 were infected with Ad5.dl802, an Ad5 adenovirus deleted for the E2A gene, at an m.o.i. of 5. Subsequently the infected cells were cultured at 32°C and cells were screened for the appearance of a cytopathic effect (CPE) as determined by changes in cell morphology and detachment of the cells from the flask.

The 293tsE2A clones c2, cl6, cl8 and c20 and the 293pcDNA3-clone c4 were tested for their E2A trans-complementing activity as follows. The cell lines were cultured in DMEM, supplemented with 10% FBS and lOmMMgCb

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PCT/NL00/00263 at 39°C and 10% CO2 in 6 wells plates and either mock infected or infected with IG.Ad.CLIP.Luc (see below) at an m.o.i. of 10. Subsequently the infected cells were cultured at either 32°C or 39°C and cells were screened for the appearance of a cytopathic effect (CPE) 3 days post infection, as determined by changes in cell morphology and detachment of the cells from the flask. Table 4 shows that no CPE appeared in the control cell line 293pcDNA3-c4. Moreover, the cell lines expressing aberrant forms of DBP either failed to complement this vector (clones 16 and 18) or were intermediate in the trans-complementing ability (clone 2). Only the 293 cell line expressing full-length tsl25E2A encoded DBP (t.e.,clone 20) fully complemented for the E2A deletion in the vector IG.Ad.CLIP.Luc at the permissive temperature of 32°C. No CPE appeared at the non-permissive temperature of 39°C.

Table 4: Complementation of E2A deletion in adenoviral vectors on 293 cells and 293 cells constitutively expressing temperature sensitive E2A.

<td> Cell line</td><td> CPE at 32°C</td><td> CPE at 39°C</td>

<td> 293pcDNA3-c4</td><td> -</td><td> -</td>

<td> 293tsl25E2A-c2</td><td> +/-</td><td> -</td>

<td> 293tsl25E2A-cl6</td><td> -</td><td> -</td>

<td> 293tsl25E2A-cl8</td><td> -</td><td> -</td>

<td> 293tsl25E2A-c20</td><td></td><td> -</td>

The 293tsl25E2A clones c2, cl6, cl8 and c20 and the 293pcDNA3-clone c4 were tested for their E2A trans-complementing activity as follows. The cell lines were either mock infected or infected with IG.Ad.CLIP.Luc at an m.o.i. of

10. Subsequently the infected cells were cultured at either 32°C or 39°C and cells were screened for the appearance of a cytopathic effect (CPE) 3 days post infection, as determined by changes in cell morphology and detachment of the cells from the flask.

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 scalable 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°C and 10% CO2 in a 175 cm² tissue culture flask (Nunc) in DMEM, supplemented with 10% FBS and lOmM MgCL. At subconfluency (70-80% confluent), the cells were washed with PBS (NPBI) and the

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PCT/NL00/00263 medium was replaced by 25 ml serum free suspension medium Ex-cell™ 525 (JRH) supplemented with 1 x L-Glutamin (Gibco BRL), hereafter designated SFM. Two days later, cells were detached from the flask by flicking and the cells were centrifuged at lOOOrpm for 5 minutes. The cell pellet was resuspended in 5 ml SFM and 0.5ml cell suspension was transferred to a 80 cm²tissue culture flask (Nunc), together with 12 ml fresh SFM. After 2 days, cells were harvested (all cells are in suspension) and counted in a Burker cell counter. Next, the cells were seeded in a 125ml 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°C in a 10% CO2 atmosphere. Cells were counted at day 1-6 in a Burker cell counter. In FIG. 4, the mean growth curve from 8 cultures is shown. PER.C6tsE2x\ c5-9 performs well in serum free suspension culture. The maximum cell density of approximately 2 x 10^G cells per ml is reached within 5 days of culture.

F. Growth characteristics of PER.C6 and PER.C6/E2A at 37°C and

39°C.

PER.C6 cells or PER.C6tsl25E2A (c8-4) cells were cultured in DMEM supplemented with 10% FBS and lOmM MgCLin a 10% CO2 atmosphere at either 37&lt;&gt;C (PER.C6) or 39°C (PER.C6tsl25E2A c8-4). At day 0, a total of 1 x 10⁶ cells were seeded per 25cm² tissue culture flask (Nunc) and the cells were cultured at the respective temperatures. At the indicated time points, cells were counted. FIG. 5 shows that the growth of PER.C6 cells at 37°C is comparable to the growth of PER.C6tsl25E2A c8-4 at 39°C. This shows that constitutive expression of tsl25E2A encoded DBP has no adverse effect on the growth of cells at the non-permissive temperature of 39°C.

G. Stability of PER.C6tsl25E2A

For several passages, the PER.C6tsl25E2A cell line clone 8-4 was cultured at 39°C and 10% CO2 in a 25 cm² tissue culture flask (Nunc) in DMEM, supplemented with 10% FBS and lOmM MgCbin the absence of selection pressure (G418). At sub-conlluency (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 ImM phenylmethylsulfonylfluoride and 0.1 mg/ml trypsin inhibitor). After 15 minutes incubation on ice, the lysates were cleared by centrifugation. Protein

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PCT/NL00/00263 concentrations were determined by the BioRad protein assay, according to standard procedures of the supplier (BioRad). Equal amounts of whole-cell extract were fractionated by SDS-PAGE on 10% gels. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6 (Reich et al., Virology 128: 480, 1983). The secondary antibody was a 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). FIG. 6 shows that the expression of tsl25E2A encoded DBP is stable for at least 16 passages, which is equivalent to approximately 40 cell doublings. No decrease in DBP levels were observed during this culture period, indicating that the expression of tsl25E2A is stable, even in the absence of G418 selection pressure.

Example II

Plasmid based system for the generation of recombinant adenoviral vectors deleted in El and E2A

A. Generation of pBr/Ad.Bam-rITR (ECACC deposit P97082122)

In order to facilitate blunt end cloning of the inverted terminal repeat (“ITR”) 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 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 GTG). After transformation into competent E.coli DH5a (Life Techn.) and analysis of ampicillin 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.

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

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PCT/NL00/00263 found to be correct. The missing G residue is complemented by the other ITR during replication.

B. Generation of pBr/Ad.Sal-rITR (ECACC deposit P97082119) pBr/Ad.Bam-rITR was digested with BamHI and Sail. The vector fragment including the adenovirus insert was isolated in 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 restriction enzyme analysis. Clone pBr/Ad.Sal-rITR contains adeno type 5 sequences from the Sail site at bp 16746 up to and including the rITR (missing the most 3’ G residue).

C. pBr/Ad.Cla-Bam (ECACC deposit P97082117) wt Adeno type 5 DNA was digested with Clal and BamHI, and the 20.6kb 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 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.Aflll-Bam (ECACC deposit P97082114)

Clone pBr/Ad.Cla-Bam was linearized with EcoRI (in pBr322) and partially digested with Aflll. After heat inactivation of Aflll for 20’ at 65°C the fragment ends were filled in with Klenow enzyme. The DNA was then ligated to a blunt double stranded oligo linker containing a Pacl site (5’AATTGTCTTAATTAACCGCTTAA-3’ (SEQ ID NO:3)). This linker was made by annealing the following two oligonucleotides: 5’AATTGTCTTAATTAACCGC-3’(SEQ ID NO:4) and 5’30 AATTGCGGTTAATTAAGAC-3’(SEQ ID NO:5), followed by blunting with Klenow enzyme. After precipitation of the ligated DNA to change buffer, the ligations were digested with an excess Pacl enzyme to remove concatamers of the oligo. The 22016 bp partial fragment containing Ado 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 Pad site and that had retained the large

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PCT/NL00/00263 adeno fragment was selected and sequenced at the 5’ end to verify correct insertion of the Pacl linker in the (lost) Aflll site.

E. Generation of pBr/Ad.Bam-rITRpac#2 (ECACC deposit P97082120) and pBr/Ad.Bam-rITR#8 (ECACC deposit P97082121)

To allow insertion of a Pad site near the ITR of Ad5 in clone pBr/Ad.Bam-rITR about 190 nucleotides were removed between the Clal site in the pBr322 backbone and the start of the ITR sequences. This was done as follows: pBr/Ad.Bam-rITR was digested with Clal and treated with nuclease Bal31 for varying lengths of time (2’, 5’, 10’ and 15’). The extend of nucleotide removal was followed by separate reactions on pBr322 DNA (also digested at the Clal site), using identical buffers and conditions. Bal31 enzyme was inactivated by incubation at 75°C for 10 minutes, the DNA was precipitated and re-suspended in a smaller volume TE buffer. To ensure blunt ends, DNA’s were further treated with T4 DNA polymerase in the presence of excess dNTPs. After digestion of the (control) pBr322 DNA with Sail, satisfactory degradation (~150 bp) was observed in the samples treated for 10’ or 15’. The 10’ or 15’ treated pBr/Ad.Bam-rITR samples were then ligated to the above described blunted Pacl linkers (See pBr/Ad.AfHI-Bam). Ligations were purified by precipitation, digested with excess Pacl and separated from the linkers on an LMP agarose gel. After re-ligation, DNA’s were transformed into competent DH5a 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, Pharmacia Biotech). Two clones were found with the Pacl linker inserted just downstream of the rITR. After digestion with Pacl, clone #2 has 28 bp and clone #8 has 27 bp attached to the ITR.

F. Generation of pWE/Ad.AflII-rITR (ECACC deposit P97082116) Cosmid vector pWE15 (Clontech) was used to clone larger Ad5 inserts.

First, a linker containing a unique Pacl site was inserted in the EcoRI sites of pWEl5 creating pWE.pac. To this end, the double stranded Pacl 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: pWE.pac digested with Pacl, pBr/Aflll-Bam digested with Pacl and BamHI and pBr/Ad.Bam-rITR#2 digested with BamHI and Pacl. These

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PCT/NL00/00263 fragments were ligated together and packaged using 1 phage packaging extracts (Stratagene) according to the manufacturer’s protocol. After infection into host bacteria, colonies were grown on plates and analyzed for presence of the complete insert. pWE/Ad.AflII-rITR contains all adenovirus type 5 sequences from bp 3534 (Aflll site) up to and including the right ITR (missing the most 3’ G residue).

G. Generation of pWE/Ad.Aflll-EcoRI pWE.pac was digested with Clal and 5’ protruding ends were filled using Klenow enzyme. The DNA was then digested with Pacl and isolated from agarose gel. pWE/Aflll-rITR was digested with EcoRI and after treatment with Klenow enzyme digested with Pacl. The large 24-kb fragment containing the adenoviral sequences was isolated from agarose gel and ligated to the Clal-digested and blunted pWE.pac vector using the Ligation Express^tm kit from Clontech. After transformation of Ultra-competent XL10-Gold cells from Stratagene, clones were identified that contained the expected insert. pWE/Aflll-EcoRI contains Ad5 sequences from bp 3534-27336.

H. Generation of pWE/Ad.AflII-rITRDE2A:

Deletion of the E2A coding sequences from pWE/Ad.AflII-rITR (ECACC deposit P97082116) has been accomplished as follows. The adenoviral sequences flanking the E2A coding region at the left and the right site were amplified from the plasmid pBr/Ad.Sal.rlTR (ECACC deposit P97082119) in a PCR reaction with the Expand PCR system (Boehringer) according to the manufacturers protocol. The following primers were used: Right flanking sequences (corresponding Ad5 nucleotides 24033 to 25180): DE2A.SnaBI: 5’GGC GTA CGT AGC CCT GTC GAA AG-3’(SEQ ID NO:6)DE2A.DBP-start: 5’CCA ATG CAT⁷ TCG AAG TAC TTC CTT CTC CTA TAG GC-3’ (SEQ ID NO:7)

The amplified DNA fragment was digested with SnaBI and Nsil (Nsil site is generated in the primer DE2A.DBP-start, underlined). In addition, a unique BstBI site is generated in this primer (italics).

Left flanking sequences (corresponding Ad5 nucleotides 21557 to 22442): DE2A.DBP-stop: 5’-CCA ATG CAT ACG GCG CAG ACG G-3’(SEQ ID NO:8)

DE2A.BamHI: 5’-GAG GTG GAT CCC ATG GAC GAG-3’(SEQ ID NO:9)

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The amplified DNA was digested with BamHI and Nsil (Nsil site is generated in the primer DE2A.DBP-stop, underlined). Subsequently, the digested DNA fragments were ligated into SnaBI/BamHI digested pBr/Ad.Sal-rlTR. Sequencing confirmed the exact replacement of the DBP coding region with a unique Nsil site and BstBI site in plasmid pBr/Ad.Sal-rITRDE2A. The unique Nsil site and BstBI site can be used to introduce an expression cassette for a gene to be transduced by the recombinant vector.

The deletion of the E2A coding sequences was performed such that the splice acceptor sites of the 100K encoding L4-gene at position 24048 in the top strand was left intact. In addition, the polyadenylation signals of the original E2A-RNA and L3-RNAs at the left hand site of the E2A coding sequences were left intact. This ensures proper expression of the L3-genes and the gene encoding the 100K L4-protein during the adenovirus life cycle.

Next, the plasmid pWE/Ad.AflII-rITRDE2A was generated. The plasmid pBr/Ad.Sal-rITRDE2A was digested with BamHI and Spel. The 3.9Kb fragment in which the E2A coding region was replaced by the unique Nsil site and BstBI site was isolated. The pWE/Ad.AflII-rITR was digested with BamHI and Spel. The 35 Kb DNA fragment, from which the BamHI/Spel fragment containing the E2A coding sequence was removed, was isolated. The fragments were ligated and packaged using 1 phage-packaging extracts according to the manufacturer protocol (Stratagene), yielding the plasmid pWE/Ad.AflII-rITRDE2A. Note that there is no sequence overlap between the adenoviral sequences present in pWE/Ad.AflII-rITRDE2A and the E2A sequences present in the expression vectors pcDNA3tsE2A and pcDNAwtE2A or the cell Unes derived from this vector.

I. Generation of the adapter plasmids

Adapter plasmid pMLP.TK (European patent application no. EP 95202213) was modified as follows: SV40 polyA sequences were amplified with primer SV40-1 (introduces a BamHI site) and SV40-2 (introduces a Bglll site).

In addition, Ad5 sequences present in this construct (from nt. 2496 to nt. 2779; Ado sequences nt. 3511 to 3794) were amplified with primers Ad5-1 (introduces a Bglll site) and Ado-2.

SV40-1: 5’-GGGGGATCCGAACTTGTTTATTGCAGC-3’ (SEQ ID NO:10).

SV40-2: 5’-GGGAGATCTAGACATGATAAGATAC-3’ (SEQ ID NO:11). Ad5-1: 5’-GGGAGATCTGTACTGAAATGTGTGGGC-3’ (SEQ ID NO:12).

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Ad5-2: 5’-GGAGGCTGCAGTCTCCAACGGCGT-3’ (SEQ ID NO:13).

Both PCR fragments were digested with Bglll and ligated. The ligation product was amplified with primers SV40-1 and Ad5-2 and digested with BamHI and AflIL The digested fragment was then ligated into pMLP.TK predigested with the same enzymes. The resulting construct, named pMLPI.TK, contains a deletion in adenovirus El sequences from nt. 459 to nt. 3510.

This plasmid was used as the starting material to make a new vector in which nucleic acid molecules comprising specific promoter and gene sequences can be easily exchanged. First, a PCR fragment was generated from pZipDMo+PyF101(N^_) template DNA (described in PCT/NL96/00195) with the following primers: LTR-1: fr-CTG TAC GTA CCA GTG CAC TGG CCT AGG CAT GGA AAA ATA CAT AAC TG-3’ (SEQ ID NO:14) and 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’ (SEQ ID NO: 15). Pwo DNA polymerase (Boehringer Mannheim) was used according to manufacturers protocol with the following temperature cycles: once 5’ at 95°C; 3’ at 55°C; and 1’ at 72°C, and 30 cycles of 1’ at 95°C, 1’ at 60°C, 1’ at 72°C, followed by once 10’ at 72°C. The PCR product was then digested with BamHI and ligated into pMLPIO (Levrero et al., 1991; Gene 101, 195-202) digested with PvuII and BamHI, thereby generating vector pLTRlO. This vector contains adenoviral sequences from bp 1 up to bp 454 followed by a promoter consisting of a part of the Mo-MuLV LTR having its wild-type enhancer sequences replaced by the enhancer from a mutant polyoma virus (PyFlOl). The promoter fragment was designated L420. Sequencing confirmed correct amplification of the LTR fragment however the most 5’ bases in the PCR fragment were missing so that the PvuII site was not restored. Next, the coding region of the murine HSA gene was inserted. pLTRlO was digested with BstBI followed by Klenow treatment and digestion with Ncol. The HSA gene was obtained by PCR amplification on pUC18-HSA (Kay et al., 1990; J. Immunol. 145, 1952-1959) using the following primers: HSA1, 5’-GCG CCA CCA TGG GCA GAG CGA TGG TGG C-3’ (SEQ ID NO:16) and HSA2, 5’-GTT AGA TCT AAG CTT GTC GAC ATC GAT CTA CTA ACA GTA GAG ATG TAG AA-3' (SEQ ID NO:17). The 269 bp-amplified fragment was sub-cloned in a shuttle vector using the Ncol and Bglll 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.

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The coding region of the HSA gene, including the TAG duplication was then excised as a Ncol (sticky)-Sall (blunt) fragment and cloned into the 3.5 kb Ncol (sticky)/BstBI (blunt) fragment from pLTRlO, resulting in pLTR-HSA10.

Finally, pLTR-HSA10 was digested with EcoRI and BamHI after which the fragment containing the left ITR, packaging signal, L420 promoter and HSA gene was inserted into vector pMLPI.TK digested with the same enzymes and thereby replacing the promoter and gene sequences. This resulted in the new adapter plasmid pAd5/L420-HSA that contains convenient recognition sites for various restriction enzymes around the promoter and gene sequences. SnaBI and Avril can be combined with Hpal, Nhel, Kpnl, Hindlll to exchange promoter sequences, while the latter sites can be combined with the Clal or BamHI sites 3’ from HSA coding region to replace genes in this construct.

Another adapter plasmid that was designed to allow easy exchange of nucleic acid molecules was made by replacing the 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 Avril and Bglll followed by treatment with Klenow to obtain blunt ends. The 5.1 kb fragment with pBr322 vector and adenoviral sequences was isolated and ligated to a blunt 1570 bp fragment from pcDNAl/amp (Invitrogen) obtained by digestion with Hhal and Avril followed by treatment with T4 DNA polymerase. This adapter plasmid was named pAd5/Clip.

The adapter plasmid pCMV.LacZ was generated as follows: The plasmid pCMV.TK (EP 95-202 213) was digested with Hindlll, blunted with Klenow and dNTPs and subsequently digested with Sail. The DNA fragment containing the CMV promoter was isolated. The plasmid pMLP.nlsLacZ (EP 95-202 213) was digested with Kpnl, blunted with T4 DNA polymerase and subsequently digested with Sail. The DNA fragment containing the LacZ gene and adjacent adenoviral sequences was isolated. Next, the two DNA fragments were ligated with T4 DNA ligase in the presence of ATP, giving rise to pCMV.nlsLacZ.

The adapter plasmid pAd5/CLIP.LacZ was generated as follows: The E.coli LacZ gene was amplified from the plasmid pMLP.nlsLacZ (EP 95202 213) by PCR with the primers

5’GGGGTGGCCAGGGTACCTCTAGGCTTTTGCAA (SEQ ID NO:18) and

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5’GGGGGGATCCATAAACAAGTTCAGAATCC (SEQ ID NO:19). The PCR reaction was performed Ex Taq (Takara) according to the suppliers protocol at the following amplification program: 5 minutes 94°C, 1 cycle; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles; 45 seconds 94°C and 30 seconds 65°C and 2 minutes 72°C, 25 cycles; 10 minutes 72; 45 seconds 94°C and 30 seconds 60°C and 2 minutes 72°C, 5 cycles, I cycle. The PCR product was subsequently digested with Kpnl and BamHI and the digested DNA fragment was ligated into KpnI/BamHI digested pcDNA3 (Invitrogen), giving rise to pcDNA3.nlsLacZ. Next, the plasmid pAd/CLIP was digested with Spel. The large fragment containing part of the 5’ part CMV promoter and the adenoviral sequences was isolated. The plasmid pcDNA3.nlsLacZ was digested with Spel and the fragment containing the 3’part of the CMV promoter and the LacZ gene was isolated. Subsequently, the fragments were ligated, giving rise to pAd/CLIP.LacZ. The reconstitution of the CMV promoter was confirmed by restriction digestion.

The adapter plasmid pAd5/CLIP.Luc was generated as follows:

The plasmid pCMV.Luc (EP 95-202 213) was digested with Hindlll and BamHI. The DNA fragment containing the luciferase gene was isolated. The adapter plasmid pAd/CLIP was digested with Hindlll and BamHI, and the large fragment was isolated. Next, the isolated DNA fragments were ligated, giving rise to pAd5/CLIP.Luc.

Example III

Generation of recombinant adenoviruses

A. El-deleted recombinant adenoviruses with wt E3 sequences

To generate El deleted recombinant adenoviruses with the plasmid-based system, the following constructs are prepared:

I. 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 adenoviral genome fragment, preferably not containing any pBr322 vector sequences, and

A complementing adenoviral genome construct pWE/Ad.Aflll-rITR digested with Pacl. These two DNA molecules are further purified by 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

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PCT/NL00/00263 adenoviruses by a one-step homologous recombination between the adapter and the complementing construct.

A general protocol as outlined hereinafter 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.AflllrlTR fragment. Adenovirus packaging cells (PER.C6) were seeded in -25 cm²flasks 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, 40μ1 lipofectamine, 4gg adapter plasmid and 4 gg of the complementing adenovirus genome fragment Aflll- rITR (or 2 gg 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 pAd/CMV-LacZ adapter. Two days later, cells are passaged to —80 cm² 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 formed. Cells and medium are harvested upon full CPE and recombinant virus is released by freeze-thawing. An extra amplification step in a 80 cm²flask is routinely performed to increase the yield since at the initial stage the titers are found to be variable despite the occurrence of full CPE. After amplification, viruses are harvested and plaque purified on 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 (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.

B. Generation of recombinant adenoviruses deleted for El and E2A

Besides replacements in the El region, it is possible to delete or replace the E2A region in the adenovirus. This 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).

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Recombinant viruses that are both El and E2A deleted are generated by a homologous recombination procedure as described above for El-replacement vectors using a plasmid-based system consisting of:

I. An adapter plasmid for El replacement according to the invention, with 5 or without insertion of a first gene of interest.

II. The pWE/Ad.AflII-rITRDE2A fragment, with or without insertion of a second gene of interest.

Generation and propagation of such viruses, e.g.,

IG.Ad.CMV.LacZDE2A, IG.Ad.CLIP.LacZDE2A, IG.Ad.CLIPDE2A or

IGAd.CLIP.LucDE2A, requires a complementing cell line for complementation of both El and E2A proteins in trans, as previously described herein.

In addition to replacements in the El and E2A region, it is also possible to delete or replace (part of) the E3 region in the El- deleted adenoviral vector, because E3 functions are not necessary for the replication, packaging and infection of the (recombinant) virus. 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 Xbal and re-ligation. This removes Ad5 wt sequences 2859220 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, 1) leaves all other coding regions intact and 2) obviates the need for a heterologous promoter since the transgene is driven by the E3 promoter and pA sequences, leaving more space for coding sequences.

To this end, the 2.7-kb EcoRI fragment from wt Ad5 containing the 5’ part of the E3 region was cloned into the EcoRI site of pBluescript (KS ) (Stratagene). Next, the Hindlll site in the polylinker was removed by digestion with EcoRV and Hindi and subsequent re-ligation. The resulting clone pBS.Eco-Eco/ad5DHIII was used to delete the gpl9K-coding region. Primers 1 (5’-GGG TAT TAG GCC AAA GGC GCA-3’ (SEQ ID NO:20)) and 2 (5’-GAT CCC ATG GAA GCT TGG GTG GCG ACC CCA GCG-3’ (SEQ ID NO:21)) were used to amplify a sequence from pBS.Eco-Eco/ad5DHIII 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’ (SEQ ID NO:22)) and 4 (5’-GTC GCT GTA GTT GGA CTG G-3’ (SEQ ID NO:23)) were used on

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PCT/NL00/00263 the same DNA to amplify Ad5 sequences from 29217 to 29476. The two resulting PCR fragments were ligated together by virtue of the new introduced Ncol site and subsequently digested with Xbal and Muni. This fragment was then ligated into the pBS.Eco-Eco/ad5DHIII vector that was digested with Xbal (partially) and Muni generating pBS.Eco-Eco/ad5DHIILDgpl9K. To allow insertion of foreign genes into the Hindlll and BamHI site, an Xbal deletion was made in pBS.Eco-Eco/ad5DHIII.Dgpl9K to remove the BamHI site in the Bluescript polylinker. The resulting plasmid pBS.EcoEco/ad5DHIIIDgpl9KDXbaI, contains unique Hindlll and BamHI sites corresponding to sequences 28733 (Hindlll) and 29218 (BamHI) in Ad5. After introduction of a foreign gene into these sites, either the deleted Xbal fragment is re-introduced, or the insert is re-cloned into pBS.EcoEco/ad5DHIII.Dgpl9K using Hindlll and for example Muni. Using this procedure, we have generated plasmids expressing HSV-TK, hIL-la, rat IL-3, luciferase or LacZ. The unique Srfl and Notl sites in the pBS.EcoEco/ad5DHIII.Dgpl9K plasmid (with or without inserted gene of interest) are used to transfer the region comprising the gene of interest into the corresponding region of pBr/Ad.Bam-rlTR, yielding construct pBr/Ad.BamrITRDgpl9K (with or without inserted gene of interest). This construct is used as described supra to produce recombinant adenoviruses. In the viral context, expression of inserted genes is driven by the adenovirus E3 promoter.

Recombinant viruses that are both El and E3 deleted are generated by a double homologous recombination procedure for Elreplacement vectors using a plasmid-based system consisting of:

I. an adapter plasmid for El replacement according to the invention, with or without insertion of a first gene of interest,

II. the pWE/Ad.Aflll-EcoRI fragment, and

III. the pBr/Ad.Bam-rITRDgpl9K plasmid with or without insertion of a second gene of interest.

In addition to manipulations in the E3 region, changes of (parts of) the E4 region can be accomplished easily in pBr/Ad.Bam-rlTR. 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 El and/or E2A and/or E4 in trans.

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Example IV

E2A revertant-free manufacturing of E1/E2A deleted vectors on PER.C6/E2A cells.

The cell lines and E1/E2A deleted vectors described hereinbefore are developed such that overlap between sequences in the recombinant adenoviral genome and E2A sequences in the complementing cell lines is avoided. This eliminates reversion of the E2A-deleted phenotype in the E1/E2A deleted recombinant adenoviral vectors due to homologous recombination. The occurrence of reversion of the E2A deleted phenotype was studied in a PCR assay.

PER.C6tsE2A clone 3-9 cells were cultured in DMEM supplemented with 10% FBS and lOmM MgCb in a 10% CO2 atmosphere at 39°C in a 25cm²tissue culture flask. At 50% confluency, cells were infected with the recombinant adenoviral vector IG.Ad.CMV.LacZDE2A and the cells were put at 32°C. Four days post infection CPE appeared and the cells were harvested by flicking the flask. Cells were pelleted by centrifugation and the cell pellet was re-suspended in 1 ml/10mM phosphate buffer (18ml 0.2M Na2HPO4 (Baker) and 7ml 0.2M NathPC^ (Merck) in 500 ml H2O pH=7.2). Next, 200 μΐ 5% sodium deoxycholate (Sigma) was added. The mixture was incubated for

30 minutes at 37°C and 50 μΐ 1M MgCla and 10μ1 DNase (IMU/ml; ICN) was added. The mixture was incubated for another half hour at 37°C and than cleared by centrifugation. The supernatant was put into a new tube and 100μ1 10% SDS (Baker) and 5μ1 Proteinase K (20mg/ml; Boehringer) were added.

The mixture was incubated for 30 minutes at 37°C and subsequently for 15 minutes at 65°C. Next, 1ml phenol (Sigma) was added and the mixture was tumbled for 1 hour and centrifuged. One ml of was supernatant was put into a fresh tube and 1 ml chloroform (Baker) was added. The mixture was tumbled for another 30 minutes and centrifuged. The supernatant was put into a fresh tube and mixed with 1 ml 2-Propanol (Baker) and the DNA was pelleted by centrifugation. The DNA was washed in 70% Ethanol (Baker) and resuspended in 200μ1 TE and Ιμΐ RNase (10 mg/ml; Boehringer). The DNA concentration was determined at a spectrophotometer.

The recombinant adenoviral vector DNA was screened for reversion of the E2A deleted phenotype by PCR. Two PCR reactions were performed (FIG.

7). The first was a nested PCR reaction for the detection of E2A sequences in the DNA sample. Two primer sets were designed. Set A contains the primers

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551: 5’CCGGCAAGTCTTGCGGCATG (SEQ ID NO:24) and 556:

5’TAGCAGGTCGGGCGCCGATAT (SEQ ID NO:25) and the nested primers 553: 5’GGCTCAGGTGGCTTTTAAGCAG (SEQ ID NO:26) and 554: 5’GAGTTGCGATACACAGGGTTGC (SEQ ID NO:27). The PCR reaction was performed using the eLONGase enzyme mix (Gibco) according to the manufacturer protocol. DNA from lxlO⁹ viral particles (+), which is equivalent to ~ 40ng, or water (-) was added as template. The PCR reactions were either not spiked, or spiked with 1, 10 and 40 molecules pBR/Ad.SalrlTR, respectively, as indicated in FIG. 7. The following amplification program for the PCR reaction with primer 551 and 556 was used: 30 seconds at 94°C, 1 cycle; 30 seconds 94°C and 30 seconds at 66°C and 90 seconds at 68°C, 35 cycles; 10 minutes 68°C, 1 cycle. One μΐ of this reaction was put into a nested PCR with primer 553 and 554 at the following amplification program: 30 seconds at 94°C, 1 cycle; 30 seconds 94°C and 30 seconds at 66°C and 90 seconds at 68°C, 35 cycles; 10 minutes 68°C, 1 cycle. This reaction yields a DNA fragment of 260 bp.

In the second PCR reaction, a set of primers (Set B) was used that flank the E2A gene in the adenoviral genome on the left and the right hand site.

This PCR reaction amplifies a DNA fragment spanning the site from which the E2A gene was deleted (FIG. 6). Primer set B comprises primer 731 5AGTGCGCAGATTAGGAGCGC (SEQ ID NO:28) and primer 734 5’TCTGCCTATAGGAGAAGGAA (SEQ ID NO:29). The PCR reaction was performed using the eLONGase enzyme mix (Gibco) according to the manufacturer protocol. DNA from lxlO⁹ viral particles (+), which is equivalent to ~ 40ng, or water (-) was added as template. The PCR reactions were either not spiked, or spiked with 1, 10 and 40 molecules pBR/Ad.SalrlTR, respectively, as indicated in FIG. 7. The following amplification program was used: 30 seconds at 94°C, 1 cycle; 30 seconds 94°C and 30 seconds at 50°C and 90 seconds at 68°C, 35 cycles; 10 minutes 68°C, 1 cycle. This PCR reaction yields a DNA fragment of 169 bp.

As shown in FIG. 7 left panel (set A), E2A sequences were amplified from the DNA samples (+) and control samples (-) spiked with both 1, 10 and 40 molecules, as evidenced by the amplification of a 260 bp DNA fragment. In contrast, no E2A sequences were amplified from the non-spiked samples. This shows that reversion of the E2A-deleted does not occur. The PCR reactions with primers 731/734 yielded the expected DNA fragment of 169 bp in the

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WO 01/07571 PCT/NLOO/00263 samples containing the recombinant adenoviral vector DNA (+). From the negative control samples containing the water instead of DNA (-), no DNA fragment of 169 bp was amplified. These data show, that elimination of overlap between adenoviral sequences in the vector and cell line prevents reversion of the E2A-deleted phenotype.

Example V

Transduction capacity of- and residual expression of adenoviral genes from Eldeleted and El/E2A-deleted recombinant adenoviral vectors.

The transduction capacity of E1/E2A deleted vectors was compared to

El deleted vectors. HeLa cells were seeded at 5χ10^δ cells/well in 6 wells plates (Greiner) in DMEM supplemented with 10% FBS in a 10% CO2 atmosphere at 37°C. The next day, cells were infected with a m.o.i. of either 0, 10, 100 or 1000 viral particles IG.Ad/CMV.LacZ or IG.Ad/CMV.LacZDE2A per cell.

Forty-eight hours post infection, cells were washed with PBS (NPBI) and fixed for 8 minutes in 0.25% glutaraldehyde (Sigma) in PBS (NPBI). Subsequently, the cells were washed twice with PBS and stained for 8 hours with X-gal solution (1 mg/ml X-gal in DMSO (Gibco), 2mM MgCh (Merck), 5mM K4[Fe(CN)e].3H2O (Merck), 5mM K3[Fe(CN)6] (Merck) in PBS. The reaction was stopped by removal of the X-gal solution and washing of the cells with PBS. FIG. 8 shows, that IG.Ad/CMV.LacZDE2A transduced HeLa cells at least as good as did IG.Ad/CMV.LacZ at all m.o.i.’s. Comparable results were obtained after infection of IG.Ad.CLIP.LacZ and IG.Ad.CLIP.LacZDE2A and after infection of A549 cells with the respective recombinant adenoviral vectors. These data show that the viral particle to transduction unit ratio (vp/tu) of E1/E2A deleted vectors (e.g., IG.Ad/CMV,LacZDE2A) is at least as good as the vp/tu of El deleted vectors (e.g., IG.Ad/CMV.LacZ).

Next, the vp/tu ratio of El- and El/E2A-deleted vectors was determined in a more sensitive assay, i.e.,a luciferase assay. HeLa and A549 cells were seeded at 5xl0⁵ cells/well in 6 wells plates (Greiner) in DMEM supplemented with 10% FBS in a 10% CO2 atmosphere at 37°C. The next day, cells were infected with a m.o.i. of either 0, 10, 100, 1,000 or 10,000 vp/cell IG.Ad/CLIP.Luc or IG.Ad/CLIP.LucDE2A per cell. Two days post infection, cells were lysed and the luciferase activity was determined with the Luciferase

Assay System according to the protocol of the supplier (Promega). FIG. 9 shows, that both the IG.Ad/CLIP.LucDE2A infected HeLa and A549 cells

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WO 01/07571 PCT/NL0Û/00263 produce more luciferase enzyme than the IG.Ad/CLIP.Luc infected HeLa and A549 and HeLa cells, at all m.o.i.’s tested. These data confirm that E1/E2A deleted recombinant adenoviral vectors produced on PER.C6tsl25E2A cells have a vp/tu ration that is at least as good as the vp/tu ratio of El deleted vectors. The above is in contrast to what has recently been reported by others (O’Neal et al., 1998; Lusky et al., 1998), who found that the vp/tu ratio of E1/E2A deleted recombinant adenoviral vectors is impaired significantly. However, these vectors were produced on two independent 293 based E2A complementing cell lines harboring inducible E2A genes. This suggests that the use of temperature sensitive E2A genes, such as tsl25E2A, yields superior E2A complementing cell lines as compared to the commonly used inducible E2A genes.

In order to test whether E1/E2A deleted vectors residually express adenoviral proteins, the following experiment has been performed. A549 cells were seeded on 6 well plates (Greiner) at a density of 5xl0⁵ cells/well in

DMEM supplemented with 10% FBS in a 10% CO2 atmosphere at 37°C. The next day, cells were infected with a m.o.i. of either 0, 100, 1,000 or 10,000 vp/cell IG.Ad/CLIP or IG.Ad.CLIPDE2xV After 12 hours, cells the infection medium was replaced by fresh DMEM supplemented with 10% FBS. Seventy20 two hours post infection, the cells were washed with PBS (NPBI) and lysed and scraped in RIPA (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in PBS, supplemented with ImM phenylmethylsulfonylfhioride 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-cell extract were fractionated by SDS-PAGE on 10% gels in triplicate. Proteins were transferred onto Immobilon-P membranes (Millipore) and incubated with the aDBP monoclonal antibody B6, the polyclonal a-Penton base antibody Ad2-Pb571 (kind gift of Dr. P. Boulanger,

Montpellier, France) and the polyclonal a-knob domain antibody of fiber

E641/3 (kind gift of R. Gerard, Leuven, Belgium). The secondary antibodies were a horseradish-peroxidase-conjugated goat anti mouse and a horseradishperoxidase-conjugated goat anti rabbit (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). FIG. 10 shows that

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WO 01/07571 PCT/NL00/00263 cells infected with IG.Ad.CLIP express both E2A encoded DBP, Penton base and Fiber proteins. The proteins co-migrated with the respective proteins in the positive control (lane P, extract from PER.C6 cells infected with IG.Ad.CLIP harvested at starting CPE). The residual expression of these proteins in A549 cells was m.o.i. dependent. In contrast, no DBP, penton-base or fiber was detected in the non-infected A549 cells or cells infected with IG.Ad.CLIPDE2A. These data show that deletion of the E2A gene did not only eliminate residual DBP expression, but also the residual expression of the late adenoviral proteins penton-base and fiber.

In conclusion, the foregoing shows that E1/E2A deleted vectors produced on PER.C6/tsE2A complementing cell lines have a favorable phenotype. First, these vectors have an ideal vp/tu ratio, which is at least as good as that of El deleted vectors. Second, the E1/E2A deleted vectors do not residuallv express detectable amounts of E2A encoded DBP or late gene encoded penton-base or fiber. This favorable phenotype improves the prospects for the use of recombinant adenoviral vectors in gene therapy.

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SEQUENCE LISTING &lt;110&gt; Fallaux, Frits J.

Hoeben, Robert C.

Bout, Abraham Valerio, Domenico van der Eb, Alex J.

Schouten, Govert J.

&lt;120&gt; MEANS AND METHODS FOR NUCLEIC ACID DELIVERY VEHICLE DESIGN AND NUCLEIC ACID TRANSFER &lt;130&gt; 3955US &lt;140&gt;

&lt;141&gt;

&lt;150&gt; US 08/793,170 &lt;151&gt; 1997-03-25 &lt;150&gt; PCT/NL96/00244 &lt;151&gt; 1996-06-14 &lt;150&gt; EP 95201728.3 &lt;151&gt; 1995-06-26 &lt;150&gt; EP 95201611.1 &lt;151&gt; 1995-06-15 &lt;160&gt; 29 &lt;170&gt; Patentln Ver. 2.0 &lt;210&gt; 1 &lt;211&gt; 35 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer DBPpcrl &lt;400&gt; 1 cgggatccgc caccatggcc agtcgggaag aggag 35 &lt;210&gt; 2 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

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WO 01/07571 PCT/NL00/00263 &lt;223&gt; Description of Artificial Sequence: primer DBPpcr2 &lt;400&gt; 2 cggaattctt aaaaatcaaa ggggttctgc cgc 33 &lt;210&gt; 3 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: oligo-linker containing Pacl site &lt;400&gt; 3 aattgtctta attaaccgct taa 23 &lt;210&gt; 4 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: oligonucleotide used to form oligo-linker described by SEQ. ID. NO.: 3 &lt;400&gt; 4 aattgtctta attaaccgc 19 &lt;210&gt; 5 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: oligonucleotide used to form oligo-linker described by SEQ. ID. NO.: 3 &lt;400&gt; 5 aattgcggtt aattaagac 19 &lt;210&gt; 6 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer DE2A.SnaBI &lt;400&gt; 6

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PCT/NLOO/00263 ggcgtacgta gccctgtcga aag 23 &lt;210&gt; 7 &lt;211&gt; 35 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer DE2A.DBP-start &lt;400&gt; 7 ccaatgcatt cgaagtactt ccttctccta taggc 35 &lt;210&gt; 8 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer DE2A.DBP-stop &lt;400&gt; 8 ccaatgcata cggcgcagac gg 22 &lt;210&gt; 9 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer DE2A.BamHI &lt;400&gt; 9 gaggtggatc ccatggacga g 21 &lt;210&gt; 10 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer SV40-1 &lt;400&gt; 10 gggggatccg aacttgttta ttgcagc &lt;210&gt; 11 &lt;211&gt; 25 &lt;212&gt; DNA

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PCT/NL00/00263 &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer SV40-2 &lt;400&gt; 11 gggagatcta gacatgataa gatac 25 &lt;210&gt; 12 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer Ad5-1 &lt;400&gt; 12 gggagatctg tactgaaatg tgtgggc 27 &lt;210&gt; 13 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer Ad5-2 &lt;400&gt; 13 ggaggctgca gtctccaacg gcgt 24 &lt;210&gt; 14 &lt;211&gt; 47 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer LTR-1 &lt;400&gt; 14 ctgtacgtac cagtgcactg gcctaggcat ggaaaaatac ataactg &lt;210&gt; 15 &lt;211&gt; 63 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer LTR-2 &lt;400&gt; 15

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WO 01/07571 gcggatcctt cgaaccatgg taagcttggt accgctagcg ttaaccgggc gactcagtca ate 63 &lt;210&gt; 16 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer HSA1 &lt;400&gt; 16 gcgccaccat gggcagagcg atggtgg 27 &lt;210&gt; 17 &lt;211&gt; 50 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer HSA2 &lt;400&gt; 17 gttagateta agettgtega catcgatcta ctaacagtag agatgtagaa 50 &lt;210&gt; 18 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer used for amplification of E coli LacZ &lt;400&gt; 18 ggggtggcca gggtacctct aggcttttgc aa 32 &lt;210&gt; 19 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer used for amplification of E coli LacZ &lt;400&gt; 19 ggggggatcc ataaacaagt tcagaatcc 29

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PCT/NL0O/00263 &lt;210&gt; 20 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 1 &lt;400&gt; 20 gggtattagg ccaaaggcgc 20 &lt;210&gt; 21 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 2 &lt;400&gt; 21 gatcccatgg aagcttgggt ggcgacccca gcg 33 &lt;210&gt; 22 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 3 &lt;400&gt; 22 gatcccatgg ggatccttta ctaagttaca aagcta 36 &lt;210&gt; 23 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 4 &lt;400&gt; 23 gtcgctgtag ttggactgg 19 &lt;210&gt; 24 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence

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PCT/NLOO/00263 &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 551 &lt;400&gt; 24 ccggcaagtcttgcggcatg 20 &lt;210&gt; 25 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 556 &lt;400&gt; 25 tagcaggtcg ggcgccgata t 21 &lt;210&gt; 26 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 553 &lt;400&gt; 26 ggctcaggtg gcttttaagc ag 22 &lt;210&gt; 27 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 554 &lt;400&gt; 27 gagttgcgat acacagggtt gc 22 &lt;210&gt; 28 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 731 &lt;400&gt; 28 agtgcgcaga ttaggagcgc 20

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PCT/NLOO/O0263 &lt;210&gt; 29 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Description of Artificial Sequence: primer 734 &lt;400&gt; 29 tctgcctata ggagaaggaa 20