Transgene Amplification and Persistence After Delivery Of

Transgene ampliﬁcation and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

Christophe Torrent,1 Carole Jullien,1 David Klatzmann,2 Michel Perricaudet,1 and Patrice Yeh1 1Aventis Pharma Gencell-Centre National de la Recherche Scientiﬁque-Institut Gustave Roussy Unite´ Mixte de Recherche 1582, Institut Gustave Roussy, 94805 Villejuif, France; 2Laboratoire de Biologie et The´rapeutique des Pathologies Immunitaires, Universite´ Pierre et Marie Curie, Centre National de la Recherche Scientiﬁque Enseignement Supe´rieur Associe´ 7087, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France.

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1␣ (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro,as well as transgene amplification and persistence in vivo. Retrovirus titers of Ͼ105 infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10- to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin. Cancer Gene Therapy (2000) 7, 1135–1144

Key words: Retrovirus; adenovirus; transgene ampliﬁcation; chimeric vector; gene therapy.

fficient delivery and stable expression of a therapeu- the difficulty in producing high-titer viral stocks for Etic gene in vivo will likely be required for several efficient in vivo delivery.2 gene therapy indications, including anti-angiogenic strat- Adenoviral vectors exhibit complementary features. egies to fight cancer and the phenotypic correction of For example, they can be grown to very high titers and many genetic deficiencies. So far, both features are not transduce many different tissues in vivo.5 First-genera- satisfactorily displayed by the vectors currently being tion vectors based on early region E1 and E3 deletions, evaluated in gene therapy protocols. however, cannot achieve stable transgene delivery and Retroviral vectors have been used successfully to expression in immunocompetent models, partly because deliver genes into a wide range of cell types in vitro as they trigger a strong cytotoxic immune response toward 1,2 well as into tissues within experimental models. Most the virally infected cells.6,7 More disabled vectors are importantly, these vectors integrate efficiently into the currently being evaluated, including the additional inac- target cell genome, and thus promote stable gene deliv- tivation of early region E4.5 For example, administration ery. Retroviral integration is precise with respect to the of E1/E4 doubly defective vectors in the livers of mice transferred information, leading to sustained transgene 3,4 immunotolerant for the transgene product has been expression. However, a significant hurdle still relies on shown to dramatically extend the survival of the virally infected cells.8,9 Transgene expression in the absence of E4, however, is impaired, especially when promoters Received November 10, 1999; accepted March 25, 2000. that respond to inflammatory factors are being used to 9–11 Address correspondence and reprint requests to Dr. Christophe Torrent, drive expression. Aventis Pharma Gencell-CNRS-IGR UMR 1582, Institut Gustave To combine the advantages of adenoviral and retro- Roussy, 94805 Villejuif, France. E-mail address: [email protected] viral vectors with regard to efficient gene delivery and

Cancer Gene Therapy, Vol 7, No 8, 2000: pp 1135–1144 1135 1136 TORRENT, JULLIEN, KLATZMANN, ET AL: ADENOVIRUS-MEDIATED RETROVIRAL DELIVERY stable genetic transduction in vivo, we have developed a generate pLTK. The SacI and SacI/NheI restriction fragments chimeric vector system in which the retroviral elements from pLTK were then cloned between the NheI and SacI sites required for recombinant retrovirus production are dis- of pCI (Promega Biotec), containing the cytomegalovirus (CMV) immediate-early enhancer/promoter, to generate played by two defective adenoviruses. Also, E1/E4 dou- Ј bly defective adenoviruses were used as carriers to take pCLTK. A NheI/NotI PCR fragment corresponding to the 3 U3 and R sequences was amplified from pLNCX with an advantage of their innocuity, a prerequisite for stable Ј Ј 8,9 upstream primer, 5 -GACCCCACCTGTAGGTTTGGC-3 , gene transfer in vivo. Herein, we report the evaluation and a downstream primer, 5Ј-AAAAAAAAGCGGCCGCTG- of this chimeric vector system in vitro and in vivo, with a CAACTGCAAGAGGG-3Ј, and cloned between the NheI and special emphasis on transgene amplification and persis- NotI sites of pCLTK to generate pCLTKR. A NotI/BamHI tence. PCR fragment corresponding to the polyadenylation signal of the hBGH gene was amplified from pcDNA3 (Invitrogen, Groningen, The Netherlands) with an upstream primer, MATERIALS AND METHODS 5Ј-AAAAAAAAGCGGCCGCCCTAAATGCTAGAGCTC- GCTG-3Ј, and a downstream primer, 5Ј-CGCGGATC- Cells CCCACCGCATCCCCAGC-3Ј, and cloned between the NotI IGRP2 cells, an E1/E4-transcomplementing cell line derived and BamHI sites of pCLTKR to generate pCLTKRpA. The from 29312 and W162 cells13 were maintained in minimum BglII/BamHI restriction fragment from pCLTKRpA contain- Eagle’s medium supplemented with 10% fetal bovine serum ing the CMV/HSV-1 tk expression cassette was inserted at the BamHI site of pXL304816 to generate pAdCLTK. The AvrII/ (FBS). Human-derived NCI-H460 non-small cell carcinoma ␣ cells (HTB177; American Type Culture Collection, Manassas, EarI fragment from pEF1 -Env, containing the env gene Va) and NIH-3T3 fibroblasts were cultivated in Dulbecco’s cassette expression, was subsequently inserted into the SalI site modified Eagle’s medium and RPMI 1640, respectively, sup- of pAdCLTK to generate the shuttle plasmid pAdCLTKEE. The shuttle plasmids pAdEGP and pAdCLTKEE were used plemented with 10% FBS. All cells were grown at 37°C in 5% ⌬ CO . to generate the corresponding E1 (nucleotides 382-3328)/ 2 ⌬E3 (nucleotides 28592–30470)/⌬E4 (nucleotides 33423– Recombinant adenoviruses 35355) adenoviral backbones, pAdGAG/POL and pAdTK/ ENV, respectively, by recombinational cloning in Escherichia The HindIII/XbaI fragment containing the human EF1␣ (elon- coli as described previously.16 Restriction analysis and partial gation factor) promoter14 was cloned between the HindIII and automated sequencing (EuroSequence Gene Service, St. Malo, XbaI of pSI (Promega Biotec, Madison, Wis) to generate France) were used to check both adenoviral backbones (Fig 1). pSI-EF1␣. The XbaI restriction fragment from pMOV-3⌬Cla (obtained from T. Heidmann, Centre National de la Recher- Adenovirus amplification and titration che Scientifique Unite´ Propre de Recherche 147, Villejuif, France), containing the Moloney murine leukemia virus The recombinant adenoviruses AdTK/ENV and AdGAG/POL (MoMLV) gag-pol genes, was cloned into the XbaI site of were recovered after transfection of IGRP2 cells with the pSI-EF1␣ to generate pEF1␣-GP. An EcoRI/BsrGI polymer- corresponding PacI-restricted pAdTK/ENV and pAdGAG/ ase chain reaction (PCR) fragment containing the gag-pol POL backbones as described previously.16 Viral prestocks were translation initiation site was amplified from pMOV-3⌬Cla aliquoted and stored at Ϫ20°C. Restriction analysis and partial with an upstream primer, 5Ј-CCGGAATTCGCCGCCACCA- automated sequencing were used to check the integrity of both TGGGCCAGACTGTTACC-3Ј, and a downstream primer, adenoviruses. 5Ј-GGAGGCGGAGGCTTAGGGTG-3Ј, and cloned between Viral titration was performed in IGRP2 cells by an immu- the EcoRI and BsrGI sites of pEF1␣-GP to generate pEF1␣- noperoxidase assay with a monoclonal antibody (Ab) specific GPaug. An XbaI/NotI PCR fragment containing the MoMLV to the Ad5 penton protein (Biodesign International, Kenne- gag-pol stop codon was amplified from pMOV-3⌬Cla with an bunkport, Me). Viral titers were expressed as infectious units upstream primer, 5Ј-GTAGACGGCATCGCAGCTTG-3Ј, (IU) per milliliter and viral particles per milliliter as deter- and a downstream primer, 5Ј-AAAAAAAAGCGGCCGCT- mined by high-performance liquid chromatography (T. Guil- CATTAGGGGGCCTCGCGGG-3Ј, and cloned between the lemin, A. Barbot, and F. Blanche, RPR-Gencell, Vitry-sur- corresponding sites of pEF1␣-GPaug to generate pEF1␣- Seine, France). GPϩ. The FspI restriction fragment from pEF1␣-GPϩ, containing the gag-pol gene cassette expression, was inserted into Adenovirus infection the EcoRV site of pXL3048 (containing the Ad5 sequence from position 1 to 382 and the pIX-encoding sequence from Subconfluent cells were infected at a known multiplicity of position 3446 to 4296) to generate the shuttle plasmid infection (MOI) in culture medium supplemented with 2% pAdEGP. FBS. After 1 hour at 37°C, cells were washed twice with An XbaI/NotI PCR fragment containing the amphotropic phosphate-buffered saline (PBS) and fresh complete medium 4070A env gene was amplified from pSV-envAM (obtained was added. For long-term analysis, infected cells were mixed at from T. Heidmann) by use of an upstream primer, 24 hours postinfection (p.i.) with noninfected cells at a 1:3 5Ј-GGCTCTAGAGCCGCCACCATGGCGCGTTCAACGC- ratio, and cultures were passed twice a week. TC-3Ј, and a downstream primer, 5Ј-AAAAAAAAGCGGC- Ј CGCTTATCATGGCTCGTACTCTATGG-3 , and cloned Recombinant retrovirus infection between the XbaI and NotI sites of pSI-EF1␣ to generate pEF1␣-Env. All retroviral infections were performed by adding diluted The BstYI/NarI fragment containing the herpes simplex virus-containing medium to the cells. One day before infection, virus-1 (HSV-1) thymidine kinase (tk) gene was cloned be- cells were seeded at 1.5 ϫ 105 cells/60-mm plate. Freshly tween the BclI and ClaI sites of retroviral vector pLNCX15 to harvested viruses were filtered (0.45-␮m pore-size filter) and

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Seine, France). The cells were washed three times with 1ϫ Power Block before being incubated with donkey anti-rabbit ﬂuorescein isothiocyanate-coupled immunoglobulin G (Jack- son ImmunoResearch Laboratories, West Grove, Penn; dilution 1/250) for 1 hour at room temperature. They were then washed three times with 1ϫ Power Block and resuspended in PBS for ﬂow cytometry analysis. Values are given as means of triplicate experiments Ϯ SE.

PCR analysis Total DNA was purified from 107 cells or tissue (50 mg) with the QIAamp blood kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The forward and reverse oligonucleotides specific for the TK provirus (the U3/TK primer set) were 5Ј-GCTAGCTTAAGTAACGCCATTT- TGC-3Ј (oligonucleotide U3) and 5Ј-GCGCCAGGT-CG- CATATCGTCGG-3Ј (oligonucleotide TK), giving rise to a 2610-bp product. PCR was performed on 2 ␮g of DNA for 40 cycles at a denaturing temperature of 95°C for 45 seconds, an annealing temperature of 62°C for 45 seconds, and an extension temperature of 72°C for 4 minutes 30 seconds. The PCR PSI/TK primer set included oligonucleotide TK (see above) and 5Ј-GCTCGTCCGGGATTTGGAGACCC-3Ј (oligonucleotide PSI), generating a 2041-bp fragment from both the TK provirus and AdTK/ENV DNA. PCR amplification was performed for 35 cycles as described above, except that the annealing temperature was 65°C. As a loading control for standardization, PCR was performed on each total DNA sample with the human ␤-actin control amplimer set (Clon- tech, Palo Alto, Calif) according to the manufacturer’s instructions. PCR products were visualized on a 1% agarose gel by ethidium bromide (EtBr) staining. PCR products were purified from the reaction mix with the QIAquick PCR Purification kit Figure 1. Molecular characterization of AdTK/ENV and AdGAG/ (Qiagen) according to the manufacturer’s instructions. POL. A: The TK, ENV, and GAG/POL expression cassettes are represented. R, U3, and U5 refer to the subsequences from the Reverse transcription (RT)-PCR analysis MoMLV LTR, respectively. ⌿ϩ refers to the extended retroviral packaging sequence. B: pAdTK/ENV and pAdGAG/POL backbones Total RNA was first extracted with the RNA-Plus kit (Quan- and the corresponding adenoviral DNAs were restricted with BglII tum Bioprobe, Illkirch, France). Polyadenylated RNAs were and run on a 0.7% agarose gel. Lanes M, 1-kb marker (Life then purified with cellulose oligo(dT) columns (Life Technol- Technologies); lanes 1 and 2, pAdTK/ENV and pAdGAG/POL back- ogies, Gaithersburg, Md) according to the manufacturer’s bone DNA, respectively; lanes 3 and 4, AdTK/ENV and AdGAG/POL instructions. RT-PCR was performed on 200 ng of mRNA with adenoviral DNA, respectively. the Access RT-PCR system (Promega Biotec) for 45 minutes at 48°C, 2 minutes at 94°C, and 40 cycles at a denaturing temperature of 94°C for 45 seconds; an annealing temperature polybrene (Sigma, St. Louis, Mo) was added at a concentration of 60°C for 45 seconds; and an extension temperature of 72°C of 8 ␮g/mL. for 3 minutes. The primers for RT-PCR were the PSI and TK To determine the titers of TK retroviral vectors, serial oligonucleotides (see above). PCR products were visualized by dilutions of viral supernatant were used to transduce NIH-3T3 EtBr staining after electrophoresis on a 0.8% agarose gel. in 6-well plates (2 ϫ 105 cells/well). Diluted supernatants were added to 2 mL of culture medium and left on the cells for 48 Quantitative slot-blot analysis hours. At this time, the percentage of TK-positive cells was ␮ ␤ determined by flow cytometry analysis (see below), and titers A total of 1 L from each PCR PSI/TK and PCR -actin were expressed as IU per milliliter. reaction mix (dilution 1, 1/10, and 1/100) were heated in 0.4 M NaOH/2.2 mM ethylenediaminetetraacetic acid at 95°C for 2 ϩ Flow cytometry analysis minutes and transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). DNA Infected cells (106) were washed with PBS, trypsinized, and was fixed by baking the membrane at 80°C for 2 hours. The fixed for 30 minutes in 4% formaldehyde-PBS at room tem- membrane was prehybridized for 2 hours at 65°C in 0.25 M perature. They were then permeabilized in 0.2% Triton X-100- NaH2PO4,0.25MNa2HPO4, and 7% sodium dodecyl sulfate PBS for 10 minutes at room temperature, washed three times (SDS), followed by overnight hybridization at 65°C in the same with 1ϫ Power Block (BioGenex Laboratories, San Ramon, buffer. It was then washed twice at 65°C in 2ϫ standard saline Calif), and incubated for 2 hours at room temperature with an citrate (SSC)/0.1% SDS for 5 minutes, twice at 65°C in 2ϫ anti-HSV-1 TK rabbit polyclonal Ab (pAbTK41, dilution SSC/0.1% SDS for 15 minutes, and twice at 65°C in 0.1ϫ 1/250; kindly provided by M. Janicot, RPR-Gencell, Vitry-sur- SSC/0.1% SDS for 30 minutes, before exposure to Fuji RX film

Cancer Gene Therapy, Vol 7, No 8, 2000 1138 TORRENT, JULLIEN, KLATZMANN, ET AL: ADENOVIRUS-MEDIATED RETROVIRAL DELIVERY at Ϫ80°C with an intensifying screen. The probe corresponded AdTK/ENV had net genome sizes of 101.4% and to the HSV-1 tk gene (AccI/PstI fragment from pLTK) that was 101.8% of the wild-type Ad5 genome, respectively. As 32 randomly labeled with [␣- P]deoxycytidine triphosphate. For checked by restriction analysis (Fig 1B) and partial the ␤-actin product, hybridization with a 30-nt oligonucleotide ␤ sequencing, the identity and integrity of both adenoviral from the human -actin gene (Clontech) was performed. genomes were confirmed. The retroviral sequences, and Membranes were exposed to Fuji imaging plates (type BAS- IIIS) and analyzed with a Fujix Bas 1000 phosphor imager with especially the repeated R sequences on both sides of the the MacBAS 2.2 program (Fuji Photo Film Co.). HSV-1 TK gene, did not recombine. Prestocks of AdTK/ ENV and AdGAG/POL were obtained with a normal ϫ 8 RT assays productivity (i.e., ranging from 3.3 to 5.8 10 IU/mL, or 1.3 to 1.9 ϫ 1010 viral particles/mL). Expression of the RT activity was quantified as described previously.17 Briefly, ␮ ␮ retroviral functions did not interfere with completion of 30- L samples mixed with 20 L of reaction cocktail were the adenoviral infectious cycle in IGRP2 cells. incubated at 37°C for 60 minutes. Samples were analyzed by autoradiography or with a Fujix Bas 1000 phosphor imager (see above). Positive controls for RT activity were Functional characterization of Ad/Rt vector system GPϩenvAm12 cell supernatants.18 To assess HSV-1 tk expression from AdTK/ENV, W162 Tumor grafting cells were infected with AdTK/ENV at an MOI of 200 IU/cell or coinfected with AdTK/ENV and AdGAG/ NCI-H460 cells were first infected in vitro at 100 IU/cell. After POL, each at an MOI of 200 IU/cell, before intracellular 18 hours, the cells were washed with PBS and harvested before ϫ 6 flow cytometry analysis with an anti-TK rabbit Ab. subcutaneous injection (5 10 cells) into the flanks of 4- to Ϯ 6-week-old female nude Swiss mice. After 23 days, the tumors Infection with AdTK/ENV yielded 67 4% of TK- were harvested and analyzed. All experiments were done in expressing cells with a mean fluorescence intensity accordance with guidelines from the French Council of Animal (MFI) of 17.5, whereas the MFI of noninfected W162 Care and Testing. cells was 1.53 (Fig 2, A and B). TK was synthesized to very similar levels in the coinfected cells, with an MFI of 16.4 (Fig 2C). Similar results were obtained when NIH- RESULTS 3T3 cells were used (data not shown). The functionality of the MoMLV gag-pol cistron was Construction of AdGAG/POL and AdTK/ENV demonstrated by an RT assay performed on culture Two E1/E3/E4-deleted recombinant backbones were supernatants of W162 cells infected with AdTK/ENV or first constructed by recombinational cloning in E. coli.16 AdGAG/POL or coinfected with AdTK/ENV and In the first backbone, the MoMLV gag-pol cistron ex- AdGAG/POL. As shown in Table 1, high levels of RT pressed from the human EF1␣ promoter has been activity were detected in AdGAG/POL-infected cell inserted in place of the E1 genes (pAdGAG/POL, see supernatants, whereas no RT activity was detectable in Materials and Methods). In the second backbone, the the medium from noninfected and AdTK/ENV-infected 4070A amphotropic env protein expressed from the cells. RT activity was also equivalent in the culture human EF1␣ promoter and the CMV/HSV-1 tk expres- medium from coinfected and AdGAG/POL-infected sion cassette containing the MoMLV PSI ϩ packaging cells. Interestingly, the RT activity from AdGAG/POL- sequence had been inserted in place of the E1 genes infected cell supernatants was 11- to 34-fold higher than (pAdTK/ENV, see Materials and Methods). that from the standard GPϩenvAm12-packaging cell With the aim to minimize the risk of recombination line.18 during backbone construction in E. coli and/or virus The infectivity of the recombinant retroviruses (i.e., amplification in IGRP2 cells, the 5Ј and 3Ј long terminal expression of a functional env gene) was demonstrated repeats (LTRs) have been engineered such that R (68 by flow cytometry quantification of TK intracellular nt) is the only region of homology left (Fig 1A). Other- levels after infection of NIH-3T3 cells with the condi- wise, the retroviral expression constructs were designed tioned medium from AdTK/ENV- and AdGAG/POL- to prevent any risk of replication-competent retrovirus coinfected W162 cells (see Materials and Methods). production. The gag-pol and env cistrons are separated Titers of Ͼ105 IU/mL were obtained, outlining the on two different adenoviruses, in which the LTRs and potential of this Ad/Rt chimeric vector system to pro- packaging signal are removed. In addition, env and duce high-titer recombinant retroviruses compared with gag-pol sequences are restricted to the open reading conventional transient methods.2,18 frames. Such features are commonly used to construct To optimize retroviral production parameters with the safe and efficient retroviral packaging cell lines.19,20 Ad/Rt vector system, W162 cells were infected with It is generally assumed that recombinant adenoviruses AdTK/ENV and AdGAG/POL at different MOIs, and resulting in viral DNA less than a net genome size of RT activity was determined after 48 hours. As shown in 105% of that of the wild-type Ad5 genome length are Table 1, there is a positive correlation between MOI and genetically stable and grow to normal titers.21 Transfec- RT activity (up to 200 IU/cell). At higher MOIs (300 and tion of the pAdGAG/POL and pAdTK/ENV backbones 500 IU/cell), there was no further increase in RT activity. in IGRP2 generated the corresponding AdGAG/POL Very similar results were recently reported by Duisit et and AdTK/ENV adenoviruses. AdGAG/POL and al22 after infection of human cells with a MoMLV

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Table 1. RT Activity

Cell supernatants MOI RT activity

Noninfected — 1 GPϩenvAm12 — 4.9 ϫ 102 AdTK/ENV 200 1 AdGAHG/POL 200 1.7 ϫ 105 AdGAG/POLϩAdTK/ENV 10/10 1.9 ϫ 104 AdGAG/POLϩAdTK/ENV 50/50 6.4 ϫ 104 AdGAG/POLϩAdTK/ENV 100/100 1.4 ϫ 105 AdGAG/POLϩAdTK/ENV 200/200 1.7 ϫ 105 AdGAG/POLϩAdTK/ENV 300/300 1.7 ϫ 105 AdGAG/POLϩAdTK/ENV 500/500 1.5 ϫ105

In vitro long-term transgene persistence and expression The persistence of the tk transgene delivered by the Ad/Rt vector system was demonstrated in a long-term in vitro assay. For this purpose, NIH-3T3 and W162 cells were used. E1/E4-deleted adenoviruses do not replicate in these two cell lines at the MOIs used (E. Vigne, unpublished observations).13 Their use herein therefore validates our data on transgene persistence (see below). NIH-3T3 and W162 cells coinfected with AdTK/ENV and AdGAG/POL (MOI of 200 IU/cell for each virus) were mixed at a 1:3 ratio with noninfected cells before being passaged twice weekly for 59 and 94 days, respectively. Noninfected cells and AdTK/ENV-infected cells (MOI of 200 IU/cell) were used in parallel as controls. Retroviral integration results in duplication, during the RT process, of the 3Ј U3 sequence to form a complete LTR (U3 R U5) at the 5Ј end of the provirus. Retrovirus-mediated integration was therefore assessed by PCR analysis of genomic DNA23–25 with a set of primers (U3/TK) speciﬁc to the TK provirus, leading to

Figure 2. TK expression analysis from AdTK/ENV-infected cells. W162 cells were infected with AdTK/ENV (200 IU/cell) (B), coinfected with AdTK/ENV plus AdGAG/POL (200 IU/cell of each virus) (C), or mock-infected (A). Intracellular levels of TK were analyzed by ﬂow cytometry with an anti-TK rabbit Ab after 48 hours. The red curve in (B) and (C) is from noninfected W162 cells. (The MFI of noninfected cells is 1.53.) Data are representative of three indepen- dent experiments. gag-pol-expressing adenovirus. Analysis of gag proteins by Western blot, with Abs recognizing the uncleaved Pr65gag precursor, indicated that the expression of this precursor saturated when high MOIs were applied on target cells. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor, affecting retroviral particle production.22 Interestingly, high RT activity persisted for Ͼ1 week Figure 3. In vitro RT activity from the chimeric Ad/RT vector system. after coinfection (Fig 3), again emphasizing the useful- W162 cells were coinfected with AdTK/ENV plus AdGAG/POL (200 ness of our hybrid system for retrovirus production IU/cell for each virus) and maintained in culture for 2 weeks without compared with standard transient procedures for which cell passages. The culture medium was changed 18 hours before RT activity rapidly declines after a 48- to 96-hour measuring the levels of RT activity (see Materials and Methods). The period.2 mean value Ϯ SE is shown. a.u., arbitrary unit.

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all timepoints tested (W162: day 20, 28, 52, and 94 p.i.; NIH-3T3: day 17 and 59 p.i.). In contrast, no TK provirus could be detected in W162 and NIH-3T3 cells infected with AdTK/ENV alone. The identity and integrity of the PCR U3/TK amplification product were confirmed by restriction analysis (Fig 4C). Proviral integration also was confirmed by Southern blot analysis of genomic DNA (XbaI-restricted) with a radiolabeled TK probe (data not shown). An oligonucleotide complementary to the MoMLV- packaging sequence (oligonucleotide PSI) also was used with the TK oligonucleotide to document transgene integration. In contrast to the U3/TK set of primers described above, the PSI/TK primer set does not differ- entiate between adenoviral AdTK/ENV DNA and the provirus sequence (Fig 5A). Despite this, no PCR product could be detected in AdTK/ENV-infected NIH- 3T3 and W162 cell cultures as early as days 17 and 20 p.i., respectively (Fig 5B), indicating that the adenoviral chromosome is rapidly lost in proliferating cells as expected (H. Leblois-Prehaud, unpublished data). This is in sharp contrast to the coinfected cells for which the cognate 2041-bp PCR product was readily amplified (Fig 5B). Again, the identity and integrity of the PCR product was assessed by restriction analysis (Fig 5C) and detection with a radiolabeled specific probe (data not shown). Consistent with these results, the presence of TK-encoding polyadenylated RNAs was demonstrated after 26 days by RT-PCR analysis with the PSI/TK set of primers in coinfected NIH-3T3 cells, but not in cells infected with AdTK/ENV alone (Fig 6). Taken together, we conclude that retrovirus-mediated transgene delivery with our chimeric vector system dramatically extends transgene persistence in proliferating cells in vitro.

Figure 4. In vitro provirus amplification. A: Representation of the TK In vivo transgene amplification provirus. R, U3, and U5 refer to the subsequences from the MoMLV LTRs, respectively. ⌿ϩ refers to the extended MoMLV packaging The functionality of the Ad/Rt vector system was further sequence. Oligonucleotides U3 and TK are separated by 2610 bp evaluated in an in vivo model. The human-derived lung and were used for genomic PCR amplification. B: PCR amplification carcinoma cell line NCI-H460 was used. E1/E4-deleted of 2 ␮g of genomic DNA from noninfected, AdTK/ENV-infected, and adenoviruses do not replicate in this cell line (data not AdTK/ENV- plus AdGAG/POL-coinfected W162 and NIH-3T3 cell shown). H460 cells were infected in vitro with a combi- cultures. Lanes M, 1-kb marker (Life Technologies); lane Ϫ, no DNA; nation of AdTK/ENV and AdGAG/POL (MOI of 100 lane ϩ, plasmid pLTK; lanes 1–3, extract from noninfected, AdTK/ IU/cell for each virus) or with AdTK/ENV alone (MOI ENV-infected, and AdTK/ENV- plus AdGAG/POL-coinfected W162 of 100 IU/cell). Flow cytometry analysis first indicated cells 94 days p.i., respectively; lanes 4–6, extract from noninfected, that these infection conditions led to ϳ25% of TK- AdTK/ENV-infected, and AdTK/ENV- plus AdGAG/POL-coinfected ϫ 6 NIH-3T3 cells 59 days p.i., respectively. C: PCR product was positive cells (data not shown). At 20 hours p.i., 5 10 purified (lane 1) before digestion with HindIII (lane 2), generating cells were implanted subcutaneously in athymic nude fragments with the expected size (1505 and 1105 bp; see (A)). mice (n ϭ 4). Noninfected cells were implanted in Lanes M, 1-kb marker (Life Technologies). PCR products were run parallel as additional control animals (n ϭ 2). Animals on a 1% agarose gel and stained with EtBr. were sacrificed at day 23 postimplantation, and tumors were harvested for analysis of transgene amplification and persistence. the amplification of a 2610-bp fragment (Fig 4A). The PCR PSI/TK analysis of tumors that developed after TK sequence from AdTK/ENV DNA cannot be ampli- coinfection with AdTK/ENV and AdGAG/POL (tumors fied with the U3/TK primer set because the U3 region G–J) indicated that they contained a higher number of tk was not included upstream of the HSV-1 tk expression gene copies compared with tumors that only received cassette (Fig 1A). As shown in Figure 4B, transgene the AdTK/ENV adenovirus (tumors C–F; Fig 7A, com- integration was detected only in cells that had been pare lanes C–F with lanes G–J) or tumors that were not coinfected with AdTK/ENV and AdGAG/POL, and at virally infected (tumors A and B). In contrast to our in

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Figure 6. RT-PCR analysis: RT-PCR ampliﬁcation of 200 ng of polyadenylated RNAs extracted from noninfected (lane 1), AdTK/ ENV-infected (lane 2), and AdTK/ENV- plus AdGAG/POL-coinfected (lane 3) NIH-3T3 cells at day 26 p.i., respectively. Lanes M, 1-kb marker (Life Technologies); lane Ϫ, no mRNA; lane ϩ, plasmid pLTK. PCR products were run on a 0.8% agarose gel and stained with EtBr.

supported a 10- to 50-fold increase in tumors cells infected with the Ad/Rt chimeric system compared with those infected with AdTK/ENV alone (Fig 7B). Integration of the tk gene was demonstrated by PCR U3/TK23–25 in all tumors derived from H460 cells infected with AdTK/ENV and AdGAG/POL (Fig 8, lanes G–J), whereas as expected, there was no evidence of TK provirus in control tumors derived from AdTK/ENV- infected cells or noninfected cells (Fig 8, lanes A–F). Proviral integration also was confirmed by Southern blot analysis of genomic DNA (XbaI restricted) with a radiolabeled TK probe (data not shown). For each of the four positive tumors (Fig 8, G–J), the PCR U3/TK signal intensity visualized by EtBr staining Figure 5. TK sequence amplification from culture cells. A: The oligonucleotides PSI and TK were used for PCR amplification of a (Fig 8) paralleled of the PCR PSI/TK signal obtained in 2041-bp fragment from AdTK/ENV DNA, as well as from the the same tumors (compare Fig 8, lanes G–J with Fig 7A, integrated TK provirus. ⌿ϩ refers to the MoMLV extended packag- lanes G–J): the PCR U3/TK and PCR PSI/TK signals ing sequence. B: PCR amplification of 2 ␮g of total DNA from were more important in tumors H and I. Taken together, noninfected, AdTK/ENV-infected, and AdTK/ENV- plus AdGAG/ these data confirmed that amplification of the tk gene POL-coinfected W162 and NIH-3T3 cell cultures. Lanes M, 1-kb copy number occurred after coinfection with AdTK/ marker (Life Technologies); lane Ϫ, no DNA; lane ϩ, plasmid pLTK; ENV and AdGAG/POL, and reflected integration of the lanes 1–3, extract from noninfected, AdTK/ENV-infected, and AdTK/ tk gene into the host chromosome in vivo. ENV- plus AdGAG/POL-coinfected W162 cells at day 20 p.i., respectively; lanes 4–6, extract from noninfected, AdTK/ENV-infected, and AdTK/ENV- plus AdGAG/POL-coinfected NIH-3T3 cells at day 17 p.i., respectively. C: PCR product was purified (lane 1) DISCUSSION before digestion with HindIII (lane 2), generating restriction fragments with the expected size (936 and 1105 bp; see (A)). PCR We reported previously that E1/E4-defective adenovi- products were run on a 1% agarose gel and stained with EtBr. ruses allow for long-term extrachromosomal persistence of the transgene in murine hepatocytes in vivo, with minimal virus-induced cytopathological effects and vec- vitro results in W162 and NIH-3T3 cells infected with tor-specific host responses.9 However, transgene expres- AdTK/ENV alone, for which no AdTK/ENV could be sion was rapidly shut down, apparently because viral detected with the PSI/TK primer set at 20 and 17 days promoters that respond to inflammatory stimuli cannot p.i., respectively (Fig 4B), a faint signal was detected be used to drive expression from this attenuated vector after 23 days in two of four NCI-H460 tumors infected context.9–11 In addition, E1/E4-deleted chromosomes with AdTK/ENV (Fig 7A, lanes C and D). A lower rate are quickly lost in proliferating tumor cells in vitro and in of division in this in vivo model compared with the W162 vivo (H. Leblois-Prehaud, unpublished observations; see and NIH-3T3 in vitro models most likely accounts for also this report). These observations prompted us to this difference. Most importantly, evaluation of the tk develop a hybrid vector system combining the high gene copy number by quantitative slot-blot analysis efficiency of delivery and long-term genome persistence

Cancer Gene Therapy, Vol 7, No 8, 2000 1142 TORRENT, JULLIEN, KLATZMANN, ET AL: ADENOVIRUS-MEDIATED RETROVIRAL DELIVERY

Figure 8. TK provirus amplification from H460 tumors. A: PCR amplification of 2 ␮g of genomic DNA from noninfected (lanes A and B), AdTK/ENV-infected (lanes C–F), and AdTK/ENV- plus AdGAG/ POL-coinfected (lanes G–J) H460 tumors. Lanes M, 1-kb marker (Life Technologies); lane Ϫ, no DNA; lane ϩ, plasmid pLTK. The oligonucleotides U3 and TK, which specifically amplify a 2610-bp fragment from the TK provirus, were used (see Fig 4A). PCR products were run on a 1% agarose gel and stained with EtBr.

coproteins, or from various mammalian species, to over- come complement-mediated inactivation of the particles in vivo.26 Indeed, several groups have used recombinant adenoviruses to facilitate the in vitro production of recombinant retroviruses with desired features. For example, first-generation (E1-defective) adenoviruses expressing the MoMLV structural genes and the vesicular stomatitis virus G-protein from a tetracycline-regulated promoter have been used to produce vesicular stomatitis virus G-protein-pseudotyped retroviral vectors.27,28 A first-generation adenovirus expressing a recombinant retroviral backbone also has been used to produce retroviruses from murine-packaging cell lines expressing Figure 7. TK sequence amplification from H460 tumors. A: PCR alternative envelopes.29 Novel retroviral producer cells ␮ amplification of 2 g of total DNA from noninfected (lanes A and B), also were developed after cotransduction with a retrovi- AdTK/ENV-infected (lanes C–F), and AdTK/ENV- plus AdGAG/POL- ral vector and a first-generation adenovirus expressing coinfected (lanes G–J) H460 tumors. Lanes M, 1-kb marker (Life 23 Technologies); lane Ϫ, no DNA; lane ϩ, plasmodia pelt. The the MoMLV genes. Finally, two E1-defective adeno- oligonucleotides PSI and TK are separated by 2041 bp and were viruses have been used successfully to deliver MoMLV- 30 used for PCR amplification (see Fig 5A). PCR products were run on based vectors. Interestingly, this chimeric vector sys- a 1% agarose gel and stained with EtBr. B: Serial dilutions (1/10) of tem allowed efficient and stable transgene delivery both the PCR amplification products were blotted onto a nylon mem- in vitro and in vivo. brane and hybridized to a radiolabeled TK probe. Serial dilutions Our data provide further characterization of Ad/Rt (1/10) of PCR ␤-actin amplification products were used as loading hybrid vectors and complement the initial reports. Our controls (see Materials and Methods). Each slot corresponds to chimeric vector system differs in several ways from the serial dilutions from a given tumor (1/1 to 1/100 from left to right). ϩ previous systems: (a) all of the retroviral functions were Lane , plasmid pLTK as positive control. Signal intensities were not incorporated within the adenovirus backbones in measured with a Fujix Bas 1000 phosphor imager. most studies;23,29 (b) the use of E1/E4-defective adenoviruses is an absolute prerequisite to extend the duration that characterize E1/E4-defective adenoviruses with the of retrovirus production by tumor-derived cells;8,9 (c) for integrative properties of recombinant retroviruses. For safety reasons, the retroviral functions were split into safety reasons, the retroviral functions were split into different adenoviral backbones; and (d) the increased two E1/E3/E4-deleted adenoviruses (AdGAG/POL and cloning capacity of our vector system should translate AdTK/ENV). Coinfected cells should thus behave as TK into higher adenovirus stability.21 retrovirus-producing cells, allowing transgene amplifica- Long-term transgene persistence (Ͼ3 months) was tion to the neighboring cells. achieved in vitro in actively dividing cells coinfected with High-titer production of infectious retrovirus was AdTK/ENV and AdGAG/POL under conditions in readily achieved in vitro, outlining the potential and which the adenoviral carrier backbones reached unde- versatility of this chimeric Ad/Rt vector system to re- tectable levels as early as 2 weeks p.i. A 10- to 50-fold cover recombinant particles with different envelope gly- transgene amplification also was demonstrated in vivo in

Cancer Gene Therapy, Vol 7, No 8, 2000 TORRENT, JULLIEN, KLATZMANN, ET AL: ADENOVIRUS-MEDIATED RETROVIRAL DELIVERY 1143 tumors coinfected with the Ad/Rt chimeric vectors com- delivery into the liver of immunocompetent mice with pared with control tumors. Retroviral integration of the E1/E4-defective adenoviruses. J Virol. 1997;71:4626– transgene was demonstrated both in vitro and in vivo 4637. with our chimeric Ad/Rt vector system. Taken together, 10. Loser P, Jennings GS, Strauss M, et al. Reactivation of the previously silenced cytomegalovirus major immediate- the integrative properties of this Ad/Rt chimeric vector ␬ system allowed for remarkable levels of transgene am- early promoter in the mouse liver: involvement of NF B. J Virol. 1998;72:180–190. plification and persistence. 11. Armentano D, Zabner J, Sacks C, et al. Effect of the E4 HIV-1-based retroviral vectors have recently shown region on the persistence of transgene expression from promise for gene transfer because they can transduce 31–33 adenovirus vectors. J Virol. 1997;71:2408–2416. nondividing cells. These vectors proved highly effi- 12. Yeh P, Dedieu JF, Orsini C, et al. Efficient dual cient for in vivo delivery and stable expression of the transcomplementation of adenovirus E1 and E4 regions 31,32,34 transgene in several target tissues. Currently, len- from a 293-derived cell line expressing a minimal E4 tivirus-derived vectors are obtained through transient functional unit. J Virol. 1996;70:559–565. transfection protocols.31,35 Adapting the Ad/Rt chimeric 13. Weinberg DH, Ketner G. A cell line that supports the vector system of this study to non-oncogenic retroviruses growth of a defective early region 4 deletion mutant of also should widen the number of clinical applications human adenovirus type 2. Proc Natl Acad Sci USA. 1983; amenable to these vectors. 80:5383–5386. 14. Mizushima S, Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990;18:5322. ACKNOWLEDGMENTS 15. Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989;7:980– We thank A. Virone and M. Janicot (RPR-Gencell, Vitry-sur- 990. Seine, France), H. Leblois-Prehaud (Unite´ Mixte de Recher- 16. Crouzet J, Naudin L, Orsini C, et al. Recombinational che (UMR) 1582 Centre National de la Recherche Scientifique construction in Escherichia coli of infectious adenoviral (CNRS), Villejuif, France), and J.-L. Salzmann (Centre Hos- genomes. Proc Natl Acad Sci USA. 1997;94:1414–1419. pitalier Universitaire, Bobigny, France) for helpful discussions 17. Goff SP, Traktman P, Baltimore D. Use of a rapid assay and advice. We thank T. Heidmann (CNRS Unite´ de Recher- for release of virion reverse transcriptase. J Virol. 1981;38: che Associeé (URA) 147, Villejuif, France) for providing 239–248. pMOV-3⌬Cla and pSV-envAM, Y. Lecluse (URA 1156 18. Markowitz D, Goff SP, Bank A. Construction and use of a CNRS, Villejuif, France) for flow cytometry analysis, F. safe and efficient amphotropic packaging cell line. Virology. Blanche and T. Guillemin (RPR-Gencell) for high-perfor- 1988;167:400–406. mance liquid chromatography support, and I. Gauffeny (UMR 19. Rigg RJ, Chen J, Dando JS, et al. A novel human 1582 CNRS, Villejuif, France) for technical support. We also amphotropic packaging cell line: high titer, complement acknowledge P. Hardouin and his staff (Institut Gustave resistance, and improved safety. Virology. 1996;218:290– Roussy, Villejuif, France) for animal care. 295. 20 Ory DS, Neugeboren BA, Mulligan RC. A stable human- derived packaging cell line for production of high titer REFERENCES retrovirus/vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci USA. 1996;93:11400–11406. 1. 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