New Tools for the Generation of E1- And/Or E3-Substituted Adenoviral Vectors

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Gene Therapy (2000) 7, 80–87  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE New tools for the generation of E1- and/or E3-substituted adenoviral vectors

X Danthinne1,2 and E Werth1 1Mountain States Medical Research Institute and Department of Veteran Affairs Medical Center, Boise, ID, USA; and 2Center for Transgene Technology and Gene Therapy, Vlaams Interuniversitair Instituut voor Biotechnologie, Leuven, Belgium

We have designed new vectors for the construction of of the sequence of the recombinant adenovirus. The recombinant adenoviruses containing expression cassettes resulting cosmid is transfected into 293 or 911 cells in order in the E1 and/or E3 regions. Using a versatile set of restric- to rescue the virus. Importantly, the method does not require tion enzymes, the cassettes are cloned into small bacterial any recombination event, either in E. coli or in mammalian vectors and subsequently introduced into large plasmids cells. The entire procedure can generate viral plaques in 12 containing the adenoviral sequences. Two positive selection days. Gene Therapy (2000) 7, 80–87. markers facilitate the recovery of a cosmid containing a copy

Keywords: adenoviral vectors; gene transfer; dicistronic vectors; adenovirus construction

Introduction mids are constructed either by recombination in E. coli11,13,14 or by in vitro ligation.15,16 The former The use of adenovirus as a vector for gene delivery is methods11,13,14 require unfortunately multiple steps in E. becoming more and more widespread. Beside the poten- coli, while the latter techniques15,16 generate the recombi- tial of adenovirus for gene therapy, it is an efficient tool nant plasmid inefficiently. The only method using to study in vitro and in vivo gene expression in cell lines cosmid technology generates virus poorly.12 or tissues that are refractory to other gene delivery In this paper we describe a quick and efficient method methods (for review, see Ref. 1). to generate adenoviral vectors that contain expression Despite recent progress to generate adenoviral vectors cassettes in early region 1 (E1) or in both early regions 1 2,3 deleted for all viral genes, E1-substituted adenoviruses and 3 (E1 and E3). Two or four cloning steps in E. coli, are still largely used. Many techniques are now available respectively, and a transfection of the resulting cosmid to construct such viruses. Most of them require recombi- into E1-expressing cells are sufficient to recover the nation between a plasmid carrying both the left end of recombinant virus. Cosmid construction and purification the adenoviral DNA and the gene of interest, and the are occasionally difficult. Therefore we use positive selec- 4 right end of the adenoviral genome, either as a linear or tion methods to facilitate the construction of the recombi- 5 circular DNA. However, these methods are time-con- nant cosmids. Furthermore, employing cosmids alleviates suming. Since the viral progeny is usually contaminated the need for extensive and time-consuming virus with the parental virus, at least two rounds of plaque purification. assays are required to obtain a pure virus preparation. Screening for the recombinant virus has been facilitated by using counter-selection methods,6–8 by extensively Results fragmenting the viral DNA complexed with the adenoviral terminal protein9 or by using Cre-lox-mediated recom- Strategy for the construction of E1-substituted bination.3 Alternative approaches have been developed adenoviruses where the sequence of the recombinant adenovirus is Our strategy to generate E1-substituted adenoviruses is 10 11–16 reconstituted either in yeast or in E. coli before being based on the construction of a cosmid vector containing 17 transfected into 293 cells. Such methods have the the sequence of the desired recombinant adenovirus. The advantage that copies of the recombinant viral DNA are advantage of using cosmid technology is that only DNAs purified from clones and should therefore generate hom- with sizes ranging between 39 and 50 kb can be packaged ogenous virus preparations. Bacterial plasmids or into ␭ particles.18 Therefore this technique will facilitate cosmids are even more attractive, since they are easily the transformation of E. coli with large plasmids and prepared in large quantities for transfections. Such plas- counter-select clones carrying small vectors that are often generated when large DNA molecules are introduced into E. coli using conventional methods.15,16 In order to Correspondence: X Danthinne, VA Medical Center, Research Service 151, facilitate cosmid construction and analysis, the gene of 500 W Fort Street, Boise, ID 83702, USA interest is linked to two positive-selection markers: the ␭ Received 23 February 1999; accepted 11 August 1999 cos site, necessary for DNA packaging into ␭ particles, E1- and/or E3-substituted adenoviral vectors X Danthinne et al 81 and the sequences coding for the lacZ ␣-peptide. The expression cassette is purified on agarose gel. This frag- presence of the cos site next to the gene of interest rather ment is ligated with either pAd242 or pAd244, both of than in the adenoviral plasmid guarantees the presence them linearized with ClaI. pAd242 is a 40.5 kb plasmid of the gene of interest in the resulting cosmid. The juxta- that contains both the remainder of the adenoviral position of the lacZ promoter/operator region next to the sequences (mu 9.2–100), with PacI and SwaI sites flanking ␣-peptide coding region ensures the correct orientation the right inverted terminal repeat (ITR), and the lacZ of the insert relative to the adenoviral sequences, by a promoter/operator sequences. pAd244 is identical to simple blue/white colony screening. pAd242, except for a 2.6 kb deletion in the E3 region. The The strategy is described in Figure 1. The first step is DNA is packaged in vitro into phage ␭ and then infected the insertion of the expression cassette into the multiple into E. coli. Because of the juxtaposition of the lacZ cloning sites of pAd063. This 3.2 kb intermediate vector promoter/operator sequences with the sequences coding contains the first 353 nucleotides of Ad5 DNA immedi- for the lacZ ␣-peptide, colonies which contain the cosmid ately preceded by PacI and SwaI sites, a ␭ cos site and the with the insert in the correct orientation will stain blue sequence coding for the lacZ ␣-peptide. These sequences in the presence of X-gal. DNA is purified, digested with are enclosed between two repeats of ClaI, BstBI and either PacIorSwaI to generate a linear adenoviral Psp1406I restriction sites, which generate in-frame com- sequence, and finally transfected into 293 or 911 cells.17,19 patible cohesive ends. The intermediate plasmid contain- Depending on the virus, the cells and the efficiency of ing the gene of interest is digested with ClaI, BstBI or transfection, plaques appear on average after 1 week but Psp1406I, whichever is not present in the expression cas- can be visible as early as 4 days after transfection. When sette, and the DNA ends are dephosphorylated using an properly organized, generation of a recombinant alkaline phosphatase. The fragment containing the ␭ cos adenovirus takes only 12 days, from the first cloning step site, the adenoviral sequences (map units 0–1) and the until plaques can be harvested. pAd242 and pAd244 have

Figure 1 Strategy for the construction of E1-substituted adenoviral vectors. Ap, ampicillin-resistance gene; Km, kanamycin-resistance gene; ori, ColE1 origin of DNA replication; lacZpo, lacZ␣: promoter/operator region and ␣-peptide sequence of E. coli lacZ gene; l-/r-ITR: left/right inverted terminal repeat; Ad5 9.2–100: Ad5 sequences from map unit 9.2 to 100.

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 82 maximal cloning capacities of 5.0 kb and 7.7 kb, respectively.

Construction of an adenovirus expressing E. coli ␤- galactosidase in the E1 region In order to demonstrate the efficiency of our method, a cassette expressing an E. coli ␤-galactosidase under the control of a human cytomegalovirus (CMV) immediate– early gene promoter and a protamine polyadenylation signal was introduced into pAd063 (Figure 2a). A BstBI fragment containing the reporter gene was purified and ligated to ClaI-digested pAd242. DNA was packaged into ␭ particles. E. coli Top10 strain (Invitrogen, Carlsbad, CA, USA) was infected and transformants were selected on Luria-Bertani (LB) medium supplemented with ampicillin, IPTG and X-gal. 193 blue colonies and 179 white colonies were obtained. Cosmid DNA was prepared from 91 blue colonies and digested with HindIII. All clones but one showed an identical restriction pattern corresponding to the insertion of the BstBI fragment in pAd242, in the correct orientation relative to the adenoviral sequence (data not shown). Six pAd242-␤Gal cosmids were purified, digested with PacI and transfected into 911 cells. On average, 12 plaques per 6 cm-diameter dish or 5 ␮g DNA were recovered. A total of 70 plaques were isolated. Virus was amplified and used to infect A549 cells. Cells infected with the various viral clones turned blue after X-gal staining, unlike non-infected cells (Figure 2c). Viral DNA was isolated from 10 clones and digested with HindIII. No difference was observed between the original cosmid DNA and the viral progeny, indicating that no gross rearrangement had occurred upon transfection (Figure 2b).

Strategy for the construction of E1- and E3-substituted adenoviruses Our system enables the construction of recombinant adenoviruses that contain two expression cassettes, one in the E1 region and the other in the E3 region, and totaling maximum 7.5 kb. As for the generation of E1- substituted viruses, cosmid technology is used. This time, each expression cassette is linked to one positive-selection marker, either the sequences coding for the lacZ ␣- peptide, or a cos site. The insertions are performed sequentially, using two intermediate vectors (Figure 3). First, the expression cassette to be inserted into the E3 region is cloned into the multiple cloning site of pAd083. This vector contains a 173 bp-long ␭ cos site, corresponding to the minimal region essential for DNA packaging into phage ␭.20 The resulting plasmid is digested with Figure 2 Construction of an adenovirus expressing E. coli ␤-galactosidase ClaI, BstBI or Psp1406I, whichever is not present in the in the E1 region. (a) Structure of cosmid pAd242-␤Gal. (b) Restriction gene of interest. The DNA ends are dephosphorylated, analysis of the genomes of 10 viral clones (Ad242-␤gal) obtained from the fragment containing the expression cassette and the pAd242-␤Gal. DNAs were digested with HindIII. As a control, cosmid ␭ cos site is purified on agarose gel and cloned into the pAd242-␤Gal was co-digested with HindIII and PacI. The lanes corre- BstBI site of pAd244. Because the ␭ cos site is linked to sponding to the molecular marker (MM) and pAd242-␤Gal have been the gene of interest, any ampicillin-resistant clone will exposed longer compared to the rest of the gel to adjust the intensities of the bands. (c) ␤-Galactosidase assay on A549 cells infected with Ad242- contain a cosmid with the expression cassette inserted ␤Gal viral clones. 3 × 104 cells were seeded in a 96-well plate and sub- into the E3 region, in either orientation relative to the sequently infected with crude viral extract. X-gal staining was performed adenoviral sequences (intermediate cosmid). A cassette 48 h later. (+) Indicates cells infected with control virus Ad259 expressing the second gene of interest is inserted into (expressing ␤-galactosidase); (−) indicates non-infected cells. another intermediate vector, pAd060. This plasmid contains the first 353 nt of Ad5 DNA flanked with PacI and SwaI sites and the sequences coding for the lacZ ␣-peptide. The resulting plasmid is digested with ClaI, BstBI

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 83

Figure 3 Strategy for the construction of recombinant adenoviral vectors, with inserts in the E1 and E3 regions. Refer to Figure 1 for abbreviations. or Psp1406I, whichever is not present in the expression transfected into 293 or 911 cells. Viral plaques appear on cassette. The DNA ends are dephosphorylated using an average 7 days after transfection. alkaline phosphatase and the fragment containing the sequences encoding the lacZ ␣-peptide, the adenoviral Construction of an adenovirus expressing Renilla and sequences and the expression cassette is purified on aga- firefly luciferases rose gel. This fragment is ligated with the intermediate In order to illustrate the efficiency of our system, a virus cosmid linearized with ClaI. The DNA is packaged in expressing both Renilla and firefly luciferases in the E1 vitro into phage ␭ and infected into E. coli. Because of the and E3 regions respectively, was constructed (Figure 4a). juxtaposition of the lacZ promoter/operator sequences First, a cassette expressing a firefly luciferase under the with the sequences coding for the lacZ ␣-peptide, colonies control of an SV40 promoter and polyadenylation signal which contain the cosmid with the insert in the correct was introduced into pAd083. A ClaI fragment containing orientation will stain blue in the presence of X-gal. The the reporter gene was purified and ligated to BstBI- resulting cosmid is purified, digested with either PacIor digested pAd244. DNA was packaged into ␭ particles SwaI to generate a linear adenoviral sequence, and finally and infected into E. coli. Cosmids from six ampicillin-

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 84 resistant clones were characterized by restriction analysis. All contained the firefly luciferase expression cassette, in either orientation (data not shown). The resulting cosmid, pAd244-FL, was then used in the second step of the procedure. A cassette expressing a Renilla luciferase under the control of an SV40 promoter and polyadenylation signal was cloned into pAd060. The resulting plasmid was digested with ClaI and the fragment containing the sequences encoding the Renilla luciferase was purified and ligated with ClaI-digested pAd244-FL. After DNA packaging into phage ␭ and E. coli infection, 135 blue colonies and 429 white colonies were obtained. Cosmids were purified from five of the blue colonies and revealed, through restriction analysis, an insert in the correct orientation relative to the adenoviral sequences (data not shown). One cosmid was further purified and digested with PacI. From two transfection experiments into 911 cells, 10 plaques were recovered, giving an average of five plaques per 6 cm-diameter dish or 5 ␮g DNA. Virus was expanded and used to infect A549 cells. After 2 days of expression, cells were lyzed and a dual luciferase assay was performed (Figure 4c). All viral clones appeared to express both luciferases. The presence of both expression cassettes in the viral genome was confirmed by restriction analysis of the viral DNAs (Figure 4b). Next, we wanted to investigate whether the presence of an expression cassette in the E3 region has an effect on viral infectivity and titer. We constructed another virus, AdM15␣, which expresses the ␤-galactosidase ⌬M15 mutant under the control of a CMV promoter and the lacZ ␣-peptide under the control of a RSV promoter, respectively, in the E1 and E3 regions. As a control, we constructed an adenovirus, AdM15, expressing the ␤- galactosidase ⌬M15 mutant under the control of a CMV promoter, in the E1 region, leaving the E3 region empty. From a single transfection experiment in 911 cells, cosmids pAdM15␣ and pAdM15 yielded eight and one plaques, respectively. For both viruses, the first plaques appeared 7 days after transfection. We prepared crude viral extracts from 293 cells infected with each of these viruses and determined the viral titers by plaque assays. It appeared that AdM15␣ grew faster and yielded a seven-fold higher titer than AdM15 (6 × 1011 p.f.u./ml versus 9 × 1010 p.f.u./ml, in triplicate experiments). These data show that the insertion of an expression cassette in the E3 region can have an effect on virus replication.

DNA yield and cosmid stability Because large plasmids are frequently unstable in E. coli and difficult to purify, we investigated the stability and yield of the adenoviral cosmids. Figure 4 Construction of an adenovirus expressing Renilla luciferase To assess the stability of our vectors, bacteria contain- (RL) in the E1 region, and firefly luciferase (FL) in the E3 region. (a) ␤ Structure of the double recombinant adenoviral cosmid pAdRL-FL. (b) ing cosmid pAd242- Gal were grown in liquid LB Restriction analysis of the genomes of 2 viral clones (AdRL-FL #1 and medium at 37°C. The culture was diluted 1000-fold each # Х 2) obtained from pAdRL-FL. Viral DNAs were co-digested with HpaI time OD600 2.0. After 48 h, the bacterial culture was and SfiI. Both 1.4 and 1.9 kb fragments (indicated by arrows on the right) purified on an agar plate. Ten clones were grown and represent the cassettes expressing Renilla and firefly luciferases, respect- revealed to contain the original cosmid as judged by ively. Control plasmids pAdRL-FL, pGL3 and pRLSV40 were digested with the same enzymes. (c) Luciferase activities from cells infected with restriction analysis. However, when the bacteria were double recombinant adenovirus AdRL-FL. A549 cells were infected with cultured extensively in the stationary phase, rearrange- the indicated viruses, at a multiplicity of infection (MOI) of 50 p.f.u. per ments were observed (data not shown). cell. Cells were lyzed 1 day after infection. RLU, arbitrary relative light Since the harvest time seemed to be important, we also units. analyzed the dependence of cosmid DNA yield on bacterial cell density. Bacteria containing cosmid pAd242- ␤Gal were grown in liquid LB at 37°C. Culture aliquots were taken every 30 min, cosmid DNA was purified,

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 85 digested with HindIII, and analyzed on agarose gel. DNA genome by recombination in E. coli or yeast represents a amounts were estimated by densitometry, after ethidium major drawback since no selection is available to guaran- bromide staining. Figure 5 shows that cosmid yield is tee an infectious DNA. Unpredicted recombination strongly dependent on the bacterial growth phase: it is events can occur, especially in yeast (X Danthinne, maximal at the end of the exponential phase and drops unpublished results), and without a thorough analysis of dramatically as the cells enter the stationary phase. Taken the recombinant plasmid or YAC, clones can be selected together, our data suggest that cosmid DNA purification that are unable to generate virus. Furthermore, recombi- should be performed using freshly grown bacteria, har- nation in E. coli requires the use of a recA+ strain, such vested at the end of the exponential growth phase. as BJ5183.11,14 The transfer of the adenoviral plasmid, product of recombination, into a recA endA strain is neces- Discussion sary to obtain high DNA yields for transfection. This additional step is not required when using the cosmid The past few years have seen the proliferation of methods approach, since the infection of E. coli with bacteriophage for the generation of recombinant adenoviral vectors. The ␭ is easily performed using recA endA strains such as method we propose in this paper has the following DH5␣, XL1-blue (Stratagene, La Jolla, CA, USA) or Top10 advantages. (Invitrogen, Carlsbad, CA, USA). Thus, using ligation to First, like several other methods,11–14 the reconstitution construct the genome of the recombinant virus in E. coli of the sequence of the desired recombinant adenovirus has strong advantages. in E. coli facilitates virus purification, since the transfec- Fourth, the proposed method allows the construction tion is performed using DNA purified from a single of double recombinant adenoviruses, containing one clone. One agar overlay on the transfected cells should expression cassette in the E1 region and the other in the therefore be sufficient to isolate a homogenous virus E3 region. With three cloning steps in E. coli and a trans- preparation. fection, our strategy is maybe no faster than other exist- Second, as has already been suggested,12 cosmid tech- ing methods,21 but probably easier since each expression nology is particularly well suited for the cloning of the cassette is inserted into the cosmid together with a posi- 36 kb-long adenoviral genome. Because of its require- tive selectable marker (either a minimal cos site, or the ment for large DNAs, the method selects clones contain- sequences encoding the lacZ ␣-peptide). So far, viruses ing full-size genomes, and excludes clones carrying small expressing two transgenes from independent promoters recombination products that are often generated when have not been used extensively,22–24 probably because of large plasmids are introduced into E. coli using conven- the difficulties in constructing them. Yet they are invalu- tional transformation methods like heat shock or able tools in case one needs to express a protein com- electroporation.15,16 In our method, we use two positive- posed of two different subunits or two proteins acting selection markers (a cos site and the lacZ sequences) to synergistically. The method we have described in this facilitate the generation of the correct cosmid. The whole paper should facilitate the construction of such viruses. procedure is very efficient, since a few hundred cosmid Fifth, the method described in this paper is very versa- clones are usually recovered. tile as far as the choice of restriction enzymes for cloning Third, our method does not require any homologous is concerned. Indeed, unlike all other existing methods, recombination event, either in E. coli,11,13,14 yeast,10 or we have flanked both adenoviral ITRs with two rare- mammalian cells.4,21 In mammalian cells, the recombi- cutting restriction enzymes (PacI and SwaI), to linearize nation step is probably rate-limiting, but the advantage the cosmid DNA before transfection. Three enzymes is that only the correct product of recombination should (ClaI, BstBI and Psp1406I) are also available to transfer generate virus. In contrast, constructing the adenoviral the expression cassettes from the intermediate plasmids (pAd063, pAd083 and pAd060) to the adenoviral plasmids (pAd242, pAd244). It is therefore likely that this method will be useful in a high number of applications. Using our vectors, the construction of an E1-substituted adenovirus will not be possible if the expression cassette contains simultaneously a ClaI, a BstBI and a Psp1406I site, or simultaneously a SwaI and a PacI site. The construction of an E1- and E3-substituted adenovirus will not be possible if the expression cassette to be inserted into the E3 region contains a ClaI site, or if the expression cassette to be inserted into the E1 region contains simultaneously a ClaI, a BstBI and a Psp1406I site, or if both SwaI and PacI sites are present in either or both expression cassettes. Finally, the method proposed in this paper is fast since E1-substituted viruses can be obtained in as little as 12 days, which is comparable to other methods currently available. The method is very reliable: in both above- described examples, 90 out of 91 cosmid clones were Figure 5 Dependence of cosmid DNA yield on bacterial growth phase. found correct by restriction analysis, all seven cosmids Bacteria carrying cosmid pAd242-␤Gal were grown in LB supplemented with 50 ␮g/ml ampicillin and aliquots were harvested every 30 min. that were transfected into 911 cells were infectious and all Cosmid DNA was purified and quantified by densitometry after HindIII plaques that were generated expressed the transgene(s). digest and agarose gel electrophoresis. A first limitation of the method is the possibility of

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 86 cosmid recombination in E. coli, simultaneously with a before reaching the stationary phase. DNA was prepared decrease of DNA yield. As shown in Figure 5, these by the alkaline lysis method,26 and further purified on potential hurdles can be avoided if the bacteria are har- a CsCl-ethidium bromide gradient or using purification vested before the cells enter the stationary growth phase. columns such as StrataPrep EF (Stratagene), Nucleobond Also, we have observed that the nature of the transgene (Clontech, San Francisco, CA, USA), or Wizard Purefec- inserted in the E1 region (pAd063-pAd242) has a slight tion (Promega, Madison, WI, USA). effect on the intensity of the blue colonies, probably because of transcriptional interference between the trans- Virus generation and analysis gene and the lacZ transcription units in E. coli (not Cosmid DNA was linearized with PacIorSwaI, ethanol- shown). precipitated and resuspended in sterile TE pH 7.5. DNA Another limitation of the technique is the size of the transfection into 911 or 293 cells, virus purification, and expression cassette that can be introduced in the adenoviral DNA characterization were performed as pre- viral genome. Our vectors allow the insertion of a viously described.27 maximum of 7.7 kb in the E1 region alone, or a total of 7.5 kb in both the E1 and E3 regions, due to the fact that Reporter gene vectors and assays the expression cassette inserted in the E3 region is A 4.3 kb cassette expressing E. coli ␤-galactosidase was flanked by a ␭ cos site. Other techniques, now available, purified by digesting plasmid pNCMVlacF (Dr RD Ger- allow the insertion of larger DNA fragments. For ard, Center for Transgene Technology and Gene Therapy, instance, up to 10 kb of transgene sequences can be intro- Leuven, Belgium) with SpeI and BglII. ␤-Galactosidase duced in the E1 region using a set of vectors deleted for assays were performed on A549 cell monolayers (ATCC the E4 region and an E4-complementing cell line.14 Also, CCL185) as previously described.28 Cassettes expressing a total of 8.1 kb of transgene sequences can be introduced firefly and Renilla luciferases were obtained, respectively, into both the E1 and E3 region, using vectors which con- from plasmids pGL3 (2.2 kb ClaI fragment) and pRLSV40 tain expanded deletions in the E1 and E3 regions.21 (1.8 kb BglII–BamHI fragment) (Promega). Dual luciferase In conclusion, we have proposed in this paper a quick assays were performed according to the protocol pro- and efficient method to generate recombinant adenoviral vided by Promega. vectors. Importantly, the method does not require any recombination event and allows the insertion of two DNA analyses expression cassettes, in the E1 and E3 regions of the aden- DNA quantifications by densitometry were carried out oviral genome. It should therefore be useful for a large using an Alpha Imager 2000 (Alpha Innotech, San Lean- number of applications. dro, CA, USA). DNA sequence analyses were performed using the GCG software package (Madison, WI, USA), Materials and methods through the Belgian EMB Node facility.

Plasmid constructions Acknowledgements The plasmids described in this paper were constructed We gratefully acknowledge Drs E Chang, Y Laroche and using PCR-based methods and many intermediate steps D Salmi for critical review of the manuscript, and Dr RE that are too long to be described here. The authors will Vestal for generous financial help. provide a detailed description upon request and will be delighted to share their vectors and start up new collab- orations with other researchers. pAd060 (Figure 3) was References constructed by replacing the ampicillin-resistance gene of 1 Robbins PD, Tahara H, Ghivizzani SC. Viral vectors for gene pUC19 with a kanamycin-resistance gene isolated from therapy. Trends Biotechnol 1998; 16: 35–40. 25 ␣ Tn903. The sequences encoding the lacZ -peptide, the 2 Kochanek S et al. A new adenoviral vector: replacement of all first 353 nucleotides from the Ad5 genome (including the viral coding sequences with 28 kb of DNA independently left ITR and the adenoviral packaging signal), and a mul- expressing both full-length dystrophin and ␤-galactosidase. Proc tiple cloning site were inserted between a tandem repeat Natl Acad Sci USA 1996; 93: 5731–5736. of ClaI, BstBI and Psp1406I sites. pAd063 (Figure 1) was 3 Hardy S et al. Construction of adenovirus vectors through constructed by inserting a ␭ cos site between the lacZ and cre-lox recombination. J Virol 1997; 71: 1842–1849. the adenoviral sequences of pAd060. pAd083 (Figure 3) 4 Kozarsky KF, Wilson JM. Gene therapy: adenovirus vectors. was made by deleting the lacZ sequences from pAd063. Curr Opin Genet Dev 1993; 3: 499–503. ␣ 5 McGrory WJ, Bautista DS, Graham FL. A simple technique for To construct pAd242 (Figure 1), the lacZ -peptide the rescue of early region I mutations into infectious human sequences of pUC19 were replaced by the right end of adenovirus type 5. Virology 1988; 163: 614–617. Ad5 genome (nt 3300-end) and a 5.7 kb DNA fragment 6 Imler JL et al. An efficient procedure to select and recover recom- from phage ␭. pAd244 (Figures 1 and 3) is a similar to binant adenovirus vectors. Gene Therapy 1995; 2: 263–268. pAd242, except that the E3 sequences corresponding to 7 Schaack J, Langer S, Guo X. Efficient selection of recombinant nt 28133–30818 in the Ad5 genome have been deleted and adenoviruses by vectors that express ␤-galactosidase. J Virol replaced with a unique BstBI site. 1995; 69: 3920–3923. 8 Davis AR, Meyers K, Wilson JM. High throughput method for creating and screening recombinant adenoviruses. Gene Therapy Cosmid constructions and purification ␭ 1998; 5: 1148–1152. Packaging of DNA into phage was performed using 9 Miyake S et al. Efficient generation of recombinant adenoviruses Gigapack III extract according to the manufacturer using adenovirus DNA–terminal protein complex and a cosmid (Stratagene). Cosmid DNA was prepared from fresh bac- bearing the full-length virus genome. Proc Natl Acad Sci USA Х teria grown in LB and harvested when OD600 2.0, ie 1996; 93: 1320–1324.

Gene Therapy E1- and/or E3-substituted adenoviral vectors X Danthinne et al 87 10 Ketner G et al. Efficient manipulation of the human adenovirus 21 Bett AJ, Haddara W, Prevec L, Graham FL. An efficient and genome as an infectious yeast artificial chromosome clone. Proc flexible system for construction of adenovirus vectors with Natl Acad Sci USA 1994; 91: 6186–6190. insertions or deletions in early regions 1 and 3. Proc Natl Acad 11 Chartier C et al. Efficient generation of recombinant adenovirus Sci USA 1994; 91: 8802–8806. vectors by homologous recombination in Escherichia coli. J Virol 22 Tomanin R et al. Development and characterization of a binary 1996; 70: 4805–4810. gene expression system based on bacteriophage T7 components 12 Fu S, Deisseroth A. Use of cosmid adenoviral vector cloning in adenovirus vectors. Gene 1997; 193: 129–140. system for the in vitro construction of recombinant adenoviral 23 Pu¨ tzer BM et al. Interleukin 12 and B7–1 costimulatory molecule vectors. Hum Gene Ther 1997; 8: 1321–1330. expressed by an adenovirus vector act synergistically to facili- 13 Crouzet J et al. Recombinational construction in Escherichia coli tate tumor regression. Proc Natl Acad Sci USA 1997; 94: 10889– of infectious adenoviral genomes. Proc Natl Acad Sci USA 1997; 10894. 94: 1414–1419. 24 Bramson J et al. Construction of a double recombinant aden- 14 He TC et al. A simplified system for generating recombinant ovirus vector expressing a heterodimeric cytokine: in vitro and Proc Natl Acad Sci USA 95 adenoviruses. 1998; : 2509–2514. in vivo production of biologically active interleukin-12. Hum 15 Mizuguchi H, Kay MA. Efficient construction of a recombinant Gene Ther 1996; 7: 333–342. adenovirus vector by an improved in vitro ligation method. Hum 25 Oka A, Sugisaki H, Takanami M. Nucleotide sequence of the Gene Ther 1999; 9: 2577–2583. kanamycin resistance transposon Tn903. J Mol Biol 1981; 147: 16 Souza DW, Armentano D. Novel cloning method for recombi- 217–226. nant adenovirus construction in Escherichia coli. Biotechniques 26 Birnboim HC, Doly J. A rapid alkaline extraction procedure for 1999; 26: 502–508. 17 Graham FL, Smiley J, Russel WC, Nairn R. Characteristics of a screening recombinant plasmid DNA. Nucleic Acids Res 1979; 7: human cell line transformed by DNA from human adenovirus 1513–1523. type 5. J Gen Virol 1977; 36: 59–74. 27 Gerard RD, Meidell RS. Adenovirus vectors. In: Hames BD, 18 Catalano CE, Cue D, Feiss M. Virus DNA packaging: the strat- Gover D (eds). DNA Cloning: A Practical Approach. Oxford Uni- egy used by phage lambda. Mol Microbiol 1995; 16: 1075–1086. versity Press: Oxford, 1995, pp 285–306. 19 Fallaux FJ et al. Characterization of 911, a new helper cell line 28 Cepko C. X-gal staining of cultured cells. In: Ausubel FM et al for the titration and propagation of early region 1-deleted aden- (eds). Current Protocols in Molecular Biology. John Wiley: New oviral vectors. Hum Gene Ther 1996; 7: 215–222. York, 1987 (Suppl. 17): pp 9.11.9–9.11.12. 20 Miwa T, Matsubara K. Lambda phage DNA sequences affecting the packaging process. Gene 1983; 24: 199–206.

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