regulation of adenovirus-mediated transgene …jvi.asm.org/content/73/10/8308.full.pdfchroboczek et...

JOURNAL OF VIROLOGY,0022-538X/99/$04.0010

Oct. 1999, p. 8308–8319 Vol. 73, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Regulation of Adenovirus-Mediated Transgene Expression bythe Viral E4 Gene Products: Requirement for E4 ORF3

M. LUSKY,* L. GRAVE, A. DIETERLE, D. DREYER, M. CHRIST, C. ZILLER, P. FURSTENBERGER,J. KINTZ, D. ALI HADJI, A. PAVIRANI, AND M. MEHTALI*

TRANSGENE S.A., 67085 Strasbourg, France

Received 3 March 1999/Accepted 12 July 1999

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2Agenes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response(Lusky et al., J. Virol. 72:2022–2032, 1998). In this study, the influence of the adenoviral E4 region on thestrength and persistence of transgene expression was evaluated by using as a model system the human cysticfibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV)promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoterin vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open readingframe 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 geneproducts but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 orE4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMVpromoter was severely reduced in the absence of E4 functions but could be partially restored in the presenceof either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of theE4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6mice are immunologically weakly responsive to the human CFTR protein. This observation implies that suchmice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

The ability of replication-deficient adenoviruses (Ad) to ef-ficiently transfer and express candidate therapeutic genes intoa variety of dividing and postmitotic cell types (4, 30, 58) makessuch viruses very effective vectors for direct in vivo gene ther-apy protocols (3, 7, 17, 19, 27, 47, 54). However, the success ofvectors defective in both E1 and E3 (AdE1°E3°) that are cur-rently used in human gene therapy is compromised by severaldrawbacks, including the demonstration that transgene expres-sion is in most cases only transient in vivo (2, 18, 21, 35, 39, 57).A series of studies in recent years have investigated the mo-lecular and immunological mechanisms involved in the in vivocontrol of transgene expression. They have suggested that thestrength and persistence of transgene expression can be influ-enced by multiple factors, such as the immunological back-ground of the selected mouse model (2, 43), the immunoge-nicity of the transgene product (43, 59, 62), the type of immuneresponse generated in the treated hosts (14), and the genomicstructure of the vector backbone (1, 10, 22). Altogether, thesestudies have established that, provided that the transgeneproduct is nonimmunogenic, recombinant Ad can allow long-term in vivo persistence of transgene expression despite theinduction of a detectable antiviral cellular and humoral im-mune response.

However, these studies also demonstrated that administra-tion of currently available Ad with deletions of E1 and E3 isoften associated with high levels of tissue toxicity and inflam-mation, which may hamper the use of such vectors at high viraldoses in human patients (19, 50, 51). To reduce the toxicity,and eventually the immunogenicity, of the recombinant vec-

tors, Ad with several regulatory genes simultaneously deletedhave been generated and analyzed for their in vitro and in vivoproperties (1, 22, 25, 26, 28, 29, 40, 45, 50, 61, 64). We haveshown in a previous study that the simultaneous deletion of theviral E1, E3, and E2A regions (AdE1°E3°E2A°) or of the E1,E3, and E4 regions (AdE1°E3°E4°) did not alter the in vivopersistence of the vector genome or affect the host cellular andhumoral antiadenovirus immune responses (40). However, thesimultaneous deletion of the E1, E3, and E4 regions did havea significant impact on in vivo liver toxicity and inflammatoryresponses. Consistent with previous reports (23, 29), we foundthat hepatotoxicity and inflammation were markedly reducedin the absence of the viral E1 and E4 regions (15). In contrast,induction of liver dystrophy and inflammation did not differwhen AdE1°E3° and AdE1°E3°E2A° vectors were compared indifferent strains of mice. Similar findings have been reportedby O’Neal et al. (50). These results imply a direct involvementof viral E4 gene products in the induction of the host inflam-matory response.

Unexpectedly, and adding to the complexity of multiple fac-tors influencing Ad-mediated transgene expression, the viralE4 region was recently shown to have a direct influence on thepersistence of transgene expression. When the transgene wasregulated from the immediate-early cytomegalovirus (CMV)promoter or the Rous sarcoma virus (RSV) promoter, long-term expression was found to be dependent on the presence ofan intact viral E4 region in cis or in trans (1, 10, 22, 31). Thesestudies suggested that the viral E4 gene products can regulate,at the transcriptional and/or posttranscriptional level, the ex-pression of nonviral genes under the control of heterologousregulatory sequences such as the CMV promoter.

To further investigate the role of the individual E4 geneproducts in the expression of CMV-driven transgenes and toaim at combining high-level and persistent transgene expres-

* Corresponding author. Mailing address: Transgene S.A., 11 rue deMolsheim, 67085 Strasbourg, France. Phone: 33 388 27 91 00. Fax: 33388 27 91 11. E-mail for M. Mehtali: [email protected]. E-mail forM. Lusky: [email protected].

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sion with low toxicity and low inflammatory response to thevector, the viral E4 region was dissected. A series of isogenicvectors carrying human cystic fibrosis transmembrane conduc-tance regulator (hCFTR) cDNA under the control of the CMVpromoter, and containing individual E4 open reading frames(ORFs) or combinations thereof, was generated. The purposeof this study was to assess the influence of the individual E4gene products on CMV-driven hCFTR transgene expression invitro and in vivo in the livers and lungs of immunocompetentand immunodeficient strains of mice.

MATERIALS AND METHODS

Viral vectors. All viral genomes were constructed as infectious plasmids byhomologous recombination in Escherichia coli as described by Chartier et al.(13). In brief, all vectors (Table 1) contain a deletion in E1 from nucleotide (nt)459 to nt 3327 and in E3 (from nt 28592 to nt 30470 or from nt 27871 to nt 30748[see Table 1]). Nucleotide numbering throughout this paper conforms to that ofChroboczek et al. (16). The E4 regions were modified as described below. Thevectors also contain in E1 the hCFTR cDNA (3) transcribed from the humanCMV promoter (6) and terminated by the polyadenylation signal from the rabbitb-globin gene.

E4 modifications. All vectors use the viral E4 promoter to drive the expressionof the wild-type or modified E4 region. The modifications introduced into the E4region of the vectors with the hCFTR expression cassette are listed in Table 1and as follows. The AdTG6418 vector (wtE4) contains the wild-type E4 region.The AdTG5643 vector (ORF1) contains a deletion in E4 removing most of theE4 coding sequences (nt 32994 to 34998) except ORF1. This deletion is identicalto the H2dl808 deletion previously described for Ad2 (12). The AdTG6447vector (ORF1-4) retains E4 ORF1, ORF3, ORF4, and ORF3/4 and lacks ORF6and ORF6/7. The deletion (from nt 32827 to nt 33985) removes the viral se-quences between the MunI (nt 32822) and AccI (nt 33984) sites. The AdTG6449vector (ORF3,4) retains E4 ORF3, ORF4, and ORF3/4. It was derived fromAdTG6447 by deletion of the viral sequences from nt 34799 to nt 35503, betweenthe PvuII site (nt 34796) and the Eco47-3 site (nt 35501). The AdTG6477 vector(ORF3) retains E4 ORF3 and was derived from AdTG6449 by a deletion withinORF4 of the sequences from nt 34069 to nt 34190, between the TthI (nt 34064)and NarI (nt 34189) sites. The AdTG6487 vector (ORF4) retains E4 ORF4 andwas derived from AdTG6447 by deletion of the sequences from ORF1 throughORF3, between the SspI site (nt 34632) and the Eco47-3 site (nt 35503). TheAdTG6421 vector (ORF6,7) retains ORF6 and ORF6/7 and was derived fromthe AdTG6418 by deletion of the sequences from ORF1 through 4, between theBglII site (nt 34112) and the AvrII site (nt 35461). The AdTG6490 vector(ORF3,6,7) retains ORF3, ORF6, and ORF6/7 and was derived from AdTG6418by deletion of the sequences from nt 34799 to nt 35503, between the PvuII site(nt 34796) and the Eco47-3 site (nt 35501). E4 ORF4 was then inactivated asdescribed above, by deletion of the sequences from nt 34069 to nt 34190, betweenthe TthI (nt 34064) and NarI (nt 34189) sites. In this construction, E4 ORF6 isnot complete: the deletion of the sequences between the TthI and NarI sites alsoremoved the first ATG codon (nt 34074) of the ORF6 sequence. Since this vectorcould be amplified to high titers in the absence of E4 complementation (see Fig.

5), we conclude that translation of the ORF6 and ORF6/7 genes starting at thesecond ATG codon (nt 34047), present at amino acid 10 in the translationalframe of ORF6, leads to functional ORF6 and ORF6/7 products (see below).

Virus generation, viral growth, and titration. For the generation of viruses, theviral genomes were released from the respective recombinant plasmids by PacIdigestion and transfected into the appropriate complementation cell lines, asdescribed previously (13, 40). Vectors with a wild-type E4 region were generatedin 293 cells, whereas all vectors with modifications of E4 were generated in293-E4ORF617 cells, described previously (40). Virus propagation, purification,and titration of infectious units (IU) by indirect immunofluorescence of the viralDNA binding protein (DBP) were carried out as described previously (40).Purified virus was stored in viral storage buffer (1 M sucrose, 10 mM Tris-HCl[pH 8.5], 1 mM MgCl2, 150 mM NaCl, 0.005% [vol/vol] Tween 80). The viralparticle concentration of each vector preparation was calculated by using theoptical density for measurement of viral DNA content (44). The particle-to-infectious-unit ratios are given in Table 1. The growth of vectors with modifica-tions of E4 in the presence and absence of E4 complementation was assessed in293 cells and compared to that in 293-E4ORF617 cells.

Animal studies. Six-week-old female immunocompetent mice (C57BL/6, H-2b;C3H, H-2k; CBA, H-2k) and immunodeficient C.B17-scid/scid mice were pur-chased from IFFA-CREDO (L’Arbreles, France). The vectors containing thehCFTR transgene were administered intratracheally, diluted in 0.9% NaCl, orintravenously, in viral storage buffer, at the indicated doses. Animals were sac-rificed at the times indicated, and organs were removed, cut into equal pieces,and immediately frozen in liquid nitrogen until analysis.

DNA analysis. Total DNA was extracted from tissue culture cells and organsas described previously (40). Briefly, cells or tissues were digested overnight witha proteinase K solution (1 mg of proteinase K in 1% sodium dodecyl sulfate[SDS]) in DNA lysis buffer (10 mM Tris-HCl [pH 7.4], 400 mM NaCl, 2 mMEDTA). Total cellular DNA was isolated by phenol-chloroform extraction fol-lowed by ethanol precipitation. DNA (10 mg) was digested with BamHI andanalyzed by Southern blot analysis using a 32P-labeled EcoRI-HindIII restrictionfragment purified from Ad5 genomic DNA (nt 27331 to 31993). The quality andquantities of DNA were monitored by ethidium bromide staining of the gelsprior to transfer.

RNA analysis. For the detection of viral gene and transgene expression, thesteady-state levels of the respective mRNAs were monitored by Northern blotanalysis. Total RNA was extracted from tissue culture cells and organs by usingthe RNA Now kit (Ozyme, Saint-Quentin-les-Yvelines, France) as recom-mended by the supplier. For Northern blot analysis, 10 to 15 mg of total RNA wassubjected to agarose gel electrophoresis (52) and transferred to nitrocellulosefilters. Filters were stained after transfer to ensure that equal amounts of totalcellular RNA were loaded and transferred. hCFTR mRNA was detected byusing a 32P-labeled BamHI restriction fragment (2,540 bp) purified from theCFTR cDNA. Viral mRNA was detected by using a 32P-labeled oligonucleotide(OTG10581), specifically hybridizing to the hexon mRNA of the viral L3 mes-sages.

Nuclear run-on transcription assay. The techniques for preparation of nucleartranscription and analysis of labeled nascent RNA by hybridization to denaturedplasmid DNAs immobilized on nitrocellulose filters were essentially as describedpreviously (32). Briefly, confluent monolayers of A549 cells were infected withthe indicated vectors at a multiplicity of infection (MOI) of 100 IU/cell andincubated for 48 h at 37°C. Nuclei from the infected cells were prepared exactlyas described previously (32). In vivo-initiated RNA transcripts from aliquotscontaining 2 3 107 nuclei were elongated in vitro for 30 min at 30°C in thepresence of 100 mCi of [a-32P]UTP (3,000 Ci/mmol) in a final volume of 200 mlcontaining 1 mg of heparin/ml, 0.6% (vol/vol) Sarkosyl, 0.4 mM (each) ATP,GTP, and CTP, 2.5 mM dithiothreitol, 0.15 mM phenylmethylsulfonyl fluoride,and 350 mM (NH4)2SO4. The reaction was terminated, and labeled RNA wasisolated, exactly as described previously (32). Dried RNA pellets were dissolvedto a final specific activity of 3.4 3 106 cpm/ml in hybridization buffer containing40% formamide, 2 mM EDTA, 0.9 M NaCl, 50 mM Na2HPO4–NaH2PO4 (pH6.5), 1% SDS, 0.4 g of polyvinylpyrrolidone/liter, 0.4 g of Ficoll/liter, 50 g ofdextran sulfate/liter, and 50 mg of denatured salmon sperm DNA/liter. A portion(1.7 3 106 cpm) of each labeled RNA was used to hybridize to immobilizeddenatured plasmid DNA containing the hCFTR cDNA, the human b-actincDNA (internal control), and the plasmid backbone (negative control). Theseplasmid DNAs were linearized, denatured in the presence of 0.3 N NaOH, andimmobilized on nitrocellulose filters by using a dot blot apparatus (5 mg ofplasmid DNA/dot). Prehybridization at 42°C for 18 h and hybridization at 42°Cfor 3 days were carried out in the same hybridization buffer (see above). Filterswere washed twice in 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate)–1% SDS for 15 min at 22°C and twice in 0.13 SSC–0.1% SDS for 15 minat 52°C. Radioactivity bound to the filter was quantified by scintillation counting.

Enzyme-linked immunospot (ELISPOT) assay. Ninety-six-well nitrocelluloseplates (Millipore, Saint Quentin les Yvelines, France) were coated with 0.3 mg ofmonoclonal rat anti-mouse gamma interferon (IFN-g) antibody (Pharmingen,Becton Dickinson, Le Pont de Claix, France)/well overnight at 4°C. Wells werewashed twice with complete Dulbecco modified Eagle medium (DMEM) plus10% fetal calf serum (FCS) and were then incubated for 2 h at 37°C with 150 mlof complete DMEM plus 10% FCS. Splenocytes recovered from CBA, C3H, orC57BL/6 mice that had been injected 7 days before sacrifice with phosphate-

TABLE 1. Properties of Ad vectors with modifications of E4

Virusa E4regionb

Promoter forhCFTR

expression

Titer(1011 IU/ml)

Titer(1012 P/ml)c

P/IUratio

AdTG6418 wt CMV 2 5.8 29AdTG5643 ORF1 CMV 1.4 3.3 23AdTG6421 ORF6,7 CMV 2.3 7.4 32AdTG6447 ORF1-4 CMV 2 3.1 16AdTG6449 ORF3,4 CMV 2.2 4.9 22AdTG6477 ORF3 CMV 1.34 2.5 18.6AdTG6487 ORF4 CMV 1 7 70AdTG6490 ORF3,6,7 CMV 3.8 7.5 20AdTG6429 wt RSV 2.3 3.9 17AdTG5687 ORF1 RSV 1.3 3.2 26

a All vectors have deletions in the viral E3 region. In AdTG6421, the deletionin E3 extends from nt 27871 to nt 30748; in all other vectors, the deletion in E3extends from nt 28592 to nt 30470. All vectors, except AdTG6429 andAdTG5687, contain the CMV-hCFTR expression cassette in place of the E1region. AdTG6429 and AdTG5687 carry the hCFTR cDNA under the control ofthe long-terminal-repeat sequences from the RSV.

b wt, wild type.c P, viral particles.

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buffered saline (PBS) or with 109 IU of an E1/E3 deletion Ad expressing or notexpressing hCFTR were plated at a concentration of 5 3 105 cells/well in avolume of 100 ml. Stimulator L929 or RMA cells infected for 6 h at an MOI of4 with a vaccinia virus expressing hCFTR were submitted to a 15-min UV-lighttreatment to inactivate the virus and were then added at a concentration of 2 3105 cells per well containing CBA/C3H or C57BL/6 splenocytes, respectively.Another stimulation was performed by direct addition of Ad vectors with orwithout the hCFTR transgene to the splenocytes at an MOI of 20. Interleukin-2(6 IU) was then added to all the wells. Plates were incubated at 37°C for 48 h andwashed five times with PBS containing 0.05% Tween 20. Wells were then incu-bated with 0.3 mg of biotinylated monoclonal rat anti-mouse IFN-g antibody(Pharmingen, Becton Dickinson)/well overnight at 4°C and subsequently werewashed five times with PBS-Tween, and 100 ml of a 1/5,000 dilution of Extravidinalkaline phosphatase (Sigma, Ivry-sur-Seine, France) was added for 45 min atroom temperature. Wells were washed five times with PBS-Tween, and 100 ml ofan alkaline phosphatase (AP) conjugate substrate kit solution (Bio-Rad, Saint-Quentin-Fallavier, France) was added for 30 min at room temperature. Thesubstrate solution was discarded, and the plates were washed under running tapwater and air dried. Colored spots were counted by using a dissecting micro-scope.

Data processing. All autoradiograms were scanned and assembled in AdobePhotoshop.

RESULTS

C57BL/6 mice develop a weak immunological response tothe hCFTR protein. To study the impact of the simultaneousdeletions of the viral E1, E3, and E4 regulatory genes on thepersistence of transgene expression, isogenic AdE1°E3°(AdTG6418) and AdE1°E3°E4° (AdTG5643) vectors carryingthe hCFTR transgene under the control of the CMV promoterwere generated (Table 1) and compared in vitro and in vivo.Since the hCFTR protein could be recognized as foreign by themouse immune system, a first series of experiments using theE1/E3 deletion vector was performed in immunodeficientSCID mice (Fig. 1A) and immunocompetent C3H, C57BL/6,

and CBA mice (Fig. 1B and C; also data not shown) to deter-mine the influence of the immune response on the persistenceof hCFTR expression. Throughout most of this study, thesteady-state level of transgene mRNA, detected by Northernblot analysis, was used to monitor the expression of the trans-gene.

As expected, intratracheal administration of AdTG6418 toimmunodeficient SCID mice led to strong and stable CMV-driven hCFTR expression in the lung, maintained over 100days (the duration of the experiment [Fig. 1A]), and the vectorgenome remained readily detectable throughout this period(Fig. 1A). In contrast, expression of hCFTR was very transientin immunocompetent C3H mice (Fig. 1B) and CBA mice (datanot shown), and the viral genome copy number declined rap-idly to undetectable levels in these animals (Fig. 1B and datanot shown). Unexpectedly, administration of the hCFTR vec-tor to the lungs of immunocompetent C57BL/6 mice resultedin stable persistence of both the viral genome and hCFTRexpression (Fig. 1C). Quantification of the Southern blots bydensitometry scanning confirmed the relatively similar persis-tence of the E1/E3 deletion vector DNA (AdTG6418) in SCIDand C57BL/6 mice and the rapid elimination of the viral ge-nome in C3H animals (Fig. 2A). We and others have previ-ously shown that C57BL/6 mice are immunologically tolerantof secreted human proteins such as coagulation factor IX (43,63, 65) or alpha-1-antitrypsin (2). These observations suggestthat C57BL/6 mice may also be tolerant of nonsecreted humanproteins such as hCFTR. To investigate this hypothesis, C3Hand C57BL/6 mice were injected intravenously with theAdTG6418 vector and their splenocytes were recovered for thedetermination of the presence of a virus- and/or transgene-

FIG. 1. Persistence of viral DNA and hCFTR gene expression in the presence of the wild-type E4 region in the lungs of immunodeficient and immunocompetentmice. The E1/E3 deletion vector expressing hCFTR (AdTG6418 [Table 1]) was administered intratracheally to SCID (A), C3H (B), and C57BL/6 (C) mice at a doseof 1.5 3 109 IU/animal, with six animals per time point for the SCID mice and five animals per time point for the C3H and C57BL/6 mice. The animals were sacrificedon the indicated days, and the persistence of the viral DNA in the lungs was analyzed by Southern blotting. Control lanes contain 10, 5, 1, and 0.1 viral genome copies,each mixed with 10 mg of lung cellular DNA from an untreated mouse (1 viral genome copy is equivalent to 30 pg of viral DNA). Expression of hCFTR in the lungwas analyzed by Northern blotting using an hCFTR-specific DNA probe. Control lanes contain 35 and 7 ng of total RNA from AdTG6418-infected 293 cells mixedwith 10 mg of lung RNA from an untreated mouse. Lung DNA and RNA were extracted and processed as described in Materials and Methods. Lanes marked n.i.contain DNA or RNA from the lungs of noninfected mice.

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specific T-cell response by an ELISPOT assay (20). This anal-ysis showed that similar antiadenoviral responses were inducedin C3H and in C57BL/6 mice treated with the E1/E3 deletionvector without any transgene (AdE1°), regardless of whetherthe splenocytes were then stimulated with the same vector orwith the hCFTR-expressing virus (Fig. 3A). However, thenumber of IFN-g-producing cells was increased in C3H mice,but not in C57BL/6 mice, when they were injected with thehCFTR-expressing vector (AdTG6418 [Table 1]) and theirsplenocytes were stimulated with AdTG6418 (Fig. 3A), sug-gesting that the anti-hCFTR response might be stronger inC3H mice than in C57BL/6 mice. This observation was con-firmed by an experiment in which the mouse splenocytes werestimulated with syngeneic L929 or RMA cells infected with avaccinia virus expressing hCFTR (Fig. 3B). Only C3H mice

were found to develop a strong cellular immune responseagainst hCFTR (Fig. 3B), confirming that C57BL/6 mice areimmunologically less responsive to hCFTR.

We conclude that a comparative in vivo evaluation ofAdE1°E3°-hCFTR (AdTG6418) and AdE1°E3°E4°-hCFTR(AdTG5643) vectors in C57BL/6 and SCID mice should allowa better determination of the influence of the viral genomicstructure on the persistence of transgene expression in bothimmunocompetent and immunodeficient hosts, without majorinterference by the anti-hCFTR immune response.

E4-mediated regulation of CMV-driven hCFTR expressionin vivo. The AdE1°E3°E4°-hCFTR vector (AdTG5643 [Table1]) was administered intratracheally and intravenously toSCID mice to determine the in vivo effect of E4 on the leveland persistence of hCFTR expression. Consistent with previ-

FIG. 2. Quantification of the persistence of the viral DNA in the presence or absence of the E4 region in the lungs of immunodeficient and immunocompetent mice.Autoradiograms corresponding to the Southern blots shown in Fig. 1 and in Fig. 4C and D were quantified by densitometry scanning, and the values are reported inpanels A and B, respectively. (A) Persistence of the viral DNA in SCID (}), C57BL/6 ( ), and C3H (F) mice injected intratracheally with the E1/E3 deletion vectorexpressing hCFTR (AdTG6418 [Table 1]). (B) Persistence of the viral DNA in C57BL/6 ( ) and C3H (F) mice injected intratracheally with the E1/E3/E4 deletionvector expressing hCFTR (AdTG5643 [Table 1]).

FIG. 3. Induction of anti-adenovirus and anti-hCFTR cellular responses in C3H and in C57BL/6 mice. Splenocytes were recovered from C3H and C57BL/6 miceinjected intravenously 7 days earlier with PBS, with an E1/E3 deletion vector expressing hCFTR (AdTG6418 [Table 1]), or with an E1/E3 deletion vector carrying notransgene (AdE1°). The splenocytes were then analyzed by an ELISPOT assay for the presence of IFN-g-expressing cells after stimulation with either AdE1° (openbars) or Ad-hCFTR (solid bars) (A) or after stimulation with syngeneic RMA cells (for C57BL/6 splenocytes) or L929 cells (for C3H splenocytes) infected with avaccinia virus expressing hCFTR (B).



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ous results (1, 10), the comparative analysis of hCFTR expres-sion in the livers (Fig. 4A) and lungs (Fig. 4B) of SCID miceshowed that transgene expression was not persistent in theabsence of the viral E4 region. Moreover, a tissue-specificmodulation of the E4 effect was apparent: in the absence of E4sequences, no expression from the CMV promoter was de-tectable in the liver at any time (Fig. 4A), while transgeneexpression in the lung, although strong early after virusadministration, declined to undetectable levels by 2 weekspostadministration (Fig. 4B). These data suggest that viral E4functions are absolutely required for the establishment andmaintenance of CMV-driven transgene expression in the liver,whereas in the lung, viral E4 functions are not required for theinitial activation of transgene expression but are required forits persistence. Thus, the viral E4 proteins, together with un-characterized tissue-specific factors, appear to control thestrength and stability of transgene expression.

A similar influence of E4 on transgene expression was ob-served in immunocompetent C57BL/6 and C3H mice injectedintratracheally with the AdE1°E3°E4°-hCFTR vector: in theabsence of E4, hCFTR was expressed in the lung at day 3postinoculation but was undetectable at day 14 in both strainsof animals (Fig. 4C and D). Quantification of the Southernblots revealed again the more-rapid decline of the viral DNAcopy number in C3H mice (Fig. 2B), probably as a conse-quence of the induction of an anti-hCFTR immune response inthese animals (Fig. 3).

Interestingly, expression from the CMV promoter is notirreversibly silenced in vivo (Fig. 5): SCID mice which wereinitially injected intratracheally with the AdE1°E3°E4°-hCFTR

vector and in which CMV-driven transgene expression haddisappeared by day 30 were reinjected by the same route at day45 with a CFTR-less AdE1°E3° vector containing E4 ORFs 1through 4. Viral DNA and hCFTR mRNA were analyzed atday 60 (15 days after administration). Figure 5 shows thatreadministration of the vector retaining E4 ORFs 1 through 4resulted in the coexistence of both viral genomes, without anysignificant change in the levels of AdE1°E4°-hCFTR vector

FIG. 4. Shutoff of CFTR expression in the absence of the viral E4 region. The E1/E3/E4 deletion vector carrying the CMV-hCFTR expression cassette (AdTG5643[Table 1]) was administered intravenously to SCID mice (A) and intratracheally to SCID (B), C3H (C), and C57BL/6 (D) mice, at a dose of 1.5 3 109 IU/animal, withfive (A) or four (B through D) animals per time point. The animals were then sacrificed on the indicated days, and the persistence of viral DNA and expression ofhCFTR in the lungs and liver were analyzed by Southern and Northern blotting, as described in the legend to Fig. 1.

FIG. 5. Reactivation of CMV-driven transgene expression by E4 gene prod-ucts. The E1/E3/E4 deletion vector carrying the CMV-hCFTR expression cas-sette (AdTG5643 [Table 1]) was administered intratracheally to SCID mice at adose of 1.5 3 109 IU/animal, with five animals per time point. Mice weresacrificed and analyzed at day 3 and day 30 postinjection. At day 45 postinjection,the mice were reinjected intratracheally with an E1/E3/E4 deletion vector withno transgene but retaining E4 ORFs 1 through 4. The presence in the lungs ofboth vector genomes and of hCFTR mRNA was analyzed as described in thelegend to Fig. 1.



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DNA. However, this administration of the vector containingE4 ORFs 1 through 4 led to a reactivation of hCFTR expres-sion by day 60. These results confirm and extend those ofBrough et al. (10) and, taken together, suggest that E4 prod-ucts encoded by ORFs 1 through 4 can in trans regulate theexpression of the CMV promoter in vivo. Moreover, these datasuggest an involvement of the E4 functions at the transcrip-tional level (see below).

Influence of the individual E4 ORFs on viral growth in vitro.The reinfection experiment described above indicated that E4sequences containing ORF1, ORF2, ORF3, ORF4, andORF3/4 are sufficient to complement in trans the deficiency ofCMV-driven transgene expression from an AdE1°E3°E4-hCFTR vector. This observation led us to investigate whetherthe E4-encoded trans-acting functions could be ascribed todefined E4 gene products.

Therefore, a series of isogenic E1/E3/E4 modification vec-tors carrying the CMV-hCFTR transgene and containing indi-vidual E4 ORFs or combinations of E4 ORFs was generated byhomologous recombination in E. coli as previously described(13) (Table 1). As shown in Fig. 6, all vectors could be purifiedto high titers in 293 cells expressing the E4 ORF6 and ORF6/7genes (38), with virus particle/infectious unit ratios below 50.The only exception was the vector containing only E4 ORF4,which reproducibly gave lower viral yields and a virus particle/infectious unit ratio greater than 50:1 (Table 1; Fig. 6). Thereduced viral yields of the AdE1°E4°ORF4 vectors in 293-E4ORF6,7 cells are consistent with the findings of a previousstudy showing strong inhibition of viral DNA replication withvectors containing ORF4 only (9). In addition, the ORF4 geneproduct has recently been reported to induce apoptosis in avariety of cell types (37, 42, 56), further supporting the negativeimpact of this protein on viral growth.

In 293 cells, only the vectors retaining ORF6 and ORF6/7,with or without the addition of ORF3 (AdTG6421 andAdTG6490 [Table 1]), could be produced to high titers (Fig.6). The vectors containing ORFs 1 through 4 (AdTG6447) orORF3, ORF4, and ORF3/4 (AdTG6449) could also replicatein the absence of E4 complementation, albeit at reduced levels(100-fold reduced compared to the vector with wild-type E4sequences). Growth of the vector retaining ORF3 only

(AdTG6477) was apparent in 293 cells, although the viralyields were reduced approximately 1,000-fold. The vectors con-taining only ORF4 (AdTG6487) or ORF1 (AdTG5643) couldnot propagate in 293 cells, as previously reported (33, 40).

It should be pointed out that the entire E4 ORF6 sequenceis included in the ORF6,7 vector (AdTG6421 [Table 1]).In contrast, in the construction of the ORF3,6,7 vector(AdTG6490 [Table 1]), the first ATG codon of ORF6 andORF6/7 has been deleted. Thus, translation of ORF6 andORF6/7 must utilize the second ATG codon (amino acid 10) inthe ORF6 translational frame. Since this vector (AdTG6490)could be produced at high titers on 293 cells (Fig. 6), the first9 N-terminal amino acids of the ORF6 and ORF6/7 geneproducts are probably dispensable, at least for viral growth.

Influence of the E4 functions on late viral and transgeneexpression in vitro. We had previously shown that the simul-taneous deletion of the viral E1, E3, and E4 regions resulted ina marked reduction of early and late viral gene expression (40).Therefore, it was of interest to monitor the impact of theindividual E4 functions on late viral gene expression. For this,noncomplementing human A549 cells were infected with theindicated vectors at a high MOI (1,000 IU/cell) and the steady-state level of mRNA encoding a representative late viral gene(hexon) was monitored by Northern blot analysis (Fig. 7A).Confirming our previous results, the AdE1°E3° vector did ex-press significant levels of hexon mRNA, although these levelswere reduced compared to the amount expressed in cells in-fected with wild-type Ad5. All other vectors showed reducedhexon mRNA expression. The two vectors retaining ORF6 andORF6/7 (AdTG6421 and AdTG6490 [Table 1]) were alsocharacterized by reduced late viral gene expression (Fig. 7A,lower panel) despite a substantial amount of viral DNA syn-thesis (Fig. 7A, upper panel). These results confirm that, in theabsence of E1 proteins, deletion of E4 ORFs impairs adeno-viral gene expression, even when cells are infected at elevatedMOIs. As previously reported (40), late viral gene expressioncould not be detected at lower MOIs, and therefore differencescould not be scored.

Transgene expression from the CMV promoter was efficientin the absence of E1 proteins when specific combinations offunctional E4 ORFs were maintained in the AdE1°E3°E4°vector (Fig. 7B). Infection of A549 cells with the indicatedvectors at an MOI of 100 IU/cell (which does not allow repli-cation of the viral DNA [Fig. 7B, upper panel]) resulted inefficient expression from the CMV promoter (Fig. 7B, lowerpanel) for vectors retaining ORFs 1 through 4 (AdTG6447) orORF3, ORF6, and ORF6/7 (AdTG6490). Weak transgene ex-pression was obtained with the vectors containing ORF3(AdTG6477) or ORF4 (AdTG6487) alone. However, trans-gene expression was significantly enhanced with the vectorretaining both ORF3 and ORF4 (AdTG6449). The vector con-taining ORF6 and ORF6/7 (AdTG6421) led to low levels oftransgene expression, but addition of ORF3 (AdTG6490)could enhance transgene expression to normal levels. In sum-mary, ORF3 together with either ORF4 or ORF6 and ORF6/7could directly or indirectly activate the CMV promoter in vitroto levels observed with the vectors containing the wild-type E4region.

The stronger activation of transgene expression in the pres-ence of ORFs 1 through 4 compared to the activation observedin the presence of ORF3 alone led us to investigate whetherORF1 could further influence the activation of the CMVpromoter. A549 cells were coinfected either with theAdE1°E4°ORF1 vector (AdTG5643) and the vector retainingORF3 alone (AdTG6477) or with AdTG6477 and the vectorretaining ORF4 alone (AdTG6487) (Fig. 7C). Such coinfec-

FIG. 6. Growth properties of the E4 modification vectors in 293 cells and in293-E4ORF6,7 cells. Cells were infected at an MOI of 2 IU/cell with E1/E3deletion vectors either retaining the wild-type (wt) E4 sequences or havingspecific modifications in the E4 region. At 48 h postinfection, the viral yields weredetermined by indirect DBP immunofluorescence (see Materials and Methods)on 293-E4ORF6,7 cells.



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tion, however, did not lead to any significant enhancement ofhCFTR expression over that seen with the vector retainingORF3 alone, suggesting a possible influence of ORF2 or of theORF3/4 splicing product. Additional viral mutants are thusneeded to further address the role of E4 ORF2 and the E4ORF3/4 splicing product in the regulation of transgene expres-sion. The influence of variable levels of expression of ORF3 inthe different vectors can, however, be excluded, since Westernblot analyses have shown that ORF3 was similarly expressed inthe wild-type E4, E4 ORF3, E4 ORF3,4, E4 ORF1-4, and E4ORF3,6,7 vectors (data not shown).

We also investigated whether the effect of the viral E4 regionwas specific to the CMV promoter or could also be observedwith other promoters. Figure 7D shows a comparison in A549cells of vectors carrying hCFTR cDNA under the control ofeither the CMV promoter (AdTG6418 and AdTG5643 [Table1]) or the RSV promoter (AdTG6429 and AdTG5687 [Table1]), both in the presence and in the absence of the viral E4region. As described above for the CMV promoter, transgeneexpression from the RSV promoter was not detectable in the

absence of the viral E4 region (Fig. 7D) (31). Whether RSVpromoter-dependent expression is similarly dependent on E4ORF3 remains to be determined. It will also be of interest toinvestigate the effect of the viral E4 region on other promoters,such as cellular promoters.

Influence of E4 functions on the rate of initiation of tran-scription from the CMV promoter. The effect of E4 on CMV-driven gene expression could be due in part to direct or indi-rect involvement of the E4 gene products in the initiation oftranscription of the transgene. To address this hypothesis, nu-clear run-on assays were performed in vitro to monitor the rateof initiation of transcription at the CMV promoter (Fig. 8).Nuclei were isolated from A549 cells infected with the indi-cated vectors and were incubated in vitro to allow previouslyinitiated RNA polymerases to elongate nascent transcripts.RNA was isolated and hybridized to denatured DNA se-quences containing human b-actin cDNA (Fig. 8A, panel 1) asan internal cellular gene control, the plasmid backbone (Fig.8A, panel 2) as a negative control, and hCFTR cDNA (Fig. 8A,panel 3). The counts bound to each probe were quantified byscintillation counting (Fig. 8B). This analysis shows that theinitiation of transcription of the hCFTR transgene was severely

FIG. 7. Expression of transgene and late viral genes in cells infected with E4modification vectors. Human A549 cells were infected with the indicated E4modification vectors at MOIs of 1,000 IU/cell (A) and 100 IU/cell (B and D). Inpanel C, the single infections were performed at an MOI of 200 IU/cell, while thedouble and triple infections were performed at an MOI of 100 IU/cell for eachvector. Wild-type (wt) Ad5 was used at an MOI of 0.5 IU/cell. Total DNA andRNA were then extracted at 72 h postinfection and processed as described in thelegend to Fig. 2 and in Materials and Methods. L3 hexon mRNA was detectedwith a 32P-labeled oligonucleotide.

FIG. 8. Transcription of the transgene in the nuclei of A549 cells infectedwith E4-modification vectors. A549 cells were infected with the indicated vectorsat an MOI of 100 IU/cell for 48 h, and nuclei were then isolated and analyzed asdescribed in Materials and Methods. (A) Denatured target DNAs were dottedon a filter and hybridized to radiolabeled RNA isolated from the nuclei of theinfected A549 cells. The target DNAs are human b-actin cDNA (panel 1), theppolyII plasmid backbone (panel 2), and hCFTR cDNA (panel 3). wt, wild type.(B) Quantification by scintillation counting of the labeled nuclear RNA hybrid-ized to the hCFTR probe dotted on the filter shown in panel A.



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reduced (30-fold) in the vector retaining E4 ORF1 only com-pared to that in the wild-type E4 vector. The presence ofORF3 alone enhanced the initiation of transcription 2- to3-fold, whereas the presence of E4 ORF3, ORF4, and ORF3/4or of ORFs 1 through 4 enhanced the initiation of hCFTRtranscription 7- and 35-fold, respectively, over that seen withthe E4 ORF1 vector. These results clearly indicate that thedifferences in steady-state mRNA levels (Fig. 7B and C) reflectdifferent transcription rates. We conclude that E4-dependenttranscriptional activation of the CMV promoter constitutespart of the mechanism by which E4 functions lead to an en-hancement of transgene expression, at least in vitro.

Influence of E4 functions on the strength and persistence oftransgene expression in vivo. To monitor the in vivo effect ofthe E4 modifications on the level and persistence of CMV-driven transgene expression, the hCFTR vectors were admin-istered by intravenous and intratracheal injections to SCIDmice. The persistence of the viral genomic DNA and transgeneexpression in the lung and liver were monitored over time bySouthern and Northern blot analysis (Fig. 9 and 10).

In the lung, the DNA profiles of all vectors were similar overtime (Fig. 9A), indicating that modifications in the vector ge-nome did not significantly affect the persistence of viral ge-nomes in vivo, as shown previously (40). Initially, all vectorsexpressed the transgene at comparable high levels (Fig. 9B).However, as observed with the AdE1°E4° vector (Fig. 4),hCFTR expression was completely shut off between day 3 andday 14 after injection with the vectors containing ORF3 alone(AdTG6477), ORF4 alone (AdTG6487), or ORF6 andORF6/7 (AdTG6421). In contrast, transgene expression per-sisted, although at different levels, with the vectors containingORFs 1 through 4 (AdTG6449), ORF3, ORF4, and ORF3/4(AdTG6447), or ORF3, ORF6, and ORF6/7 (AdTG6490).Interestingly, the presence of ORF3 together with ORF6 andORF7 (AdTG6490) led to constitutive expression at levelshigher than those in the presence of both ORF3 and ORF4(AdTG6447) or in the presence of the wild-type E4 region(AdTG6418). CMV-driven expression from the vectors retain-ing ORFs 1 through 4 (AdTG6449) or ORF3, ORF4, and

ORF3/4 (AdTG6447) appeared reduced at day 14 postinjec-tion, followed by an induction at day 45 and a decline again atday 83. This was not due to differential recovery of RNA, sincestaining of the nitrocellulose filters after RNA transfer re-vealed that equivalent amounts of total mRNA were loadedand transferred (data not shown). These results indicate that inthe lung, the initial establishment of transgene expression fromthe CMV promoter is independent of any viral E4 function.However, maintaining strong and persistent transgene expres-sion absolutely requires E4 ORF3 in conjunction with eitherORF4 or ORF6 and ORF6/7.

In the liver, the persistence of transgene expression was alsodependent on E4 ORF3 function (Fig. 10; also data notshown). However, in comparison to the pattern of transgeneexpression in the lung, two major differences were noticed.First, the initial activation of the CMV promoter in the liverwas absolutely dependent on the ORF3 gene product. Notransgene expression was observed at day 3 after administra-tion of vectors lacking ORF3 (Fig. 10; also data not shown).Second, E4 ORF3 alone was sufficient for persistent expres-sion from the CMV promoter; sustained expression ofhCFTR did not require the cooperation of ORF3 with eitherORF4 or ORF6 and ORF6/7. However, the activation ofE3-dependent transgene expression was delayed with theAdE1°E3°E4°ORF3-hCFTR vector. This delay in the appear-ance of CFTR mRNA could be overcome in the presence ofeither ORF4 or ORF6 and ORF6/7 in addition to ORF3. Thefact that E4 ORF3 was sufficient for long-term transgene ex-pression was confirmed with different transgenes (41).

Together, our results support the notion that the status ofthe viral E4 region influences the strength and persistence oftransgene expression (1, 10), with ORF3 playing a pivotal rolein this genetic regulation. While ORF3 is sufficient in the liver,additional viral factors, such as E4 ORF4 or ORF6 andORF6/7 and/or tissue-specific factors, appear to be requiredfor stable transgene expression in the lung. Furthermore, theE4-dependent enhancement of gene expression is at least inpart due to activation of the CMV promoter at the level of theinitiation of transcription.

FIG. 9. E4 ORF3 is required but not sufficient for the persistence of CMV-CFTR expression in the lungs of SCID mice. Vectors with wild-type (wt) E4 sequencesor with specific modifications in the E4 region were administered by intratracheal injection to SCID mice at a dose of 1.5 3 109 IU/animal, with four animals per timepoint. Animals were sacrificed on the indicated days. The persistence of viral DNA (A) and of hCFTR expression (B) in the lung were determined by Southern andNorthern blot analysis, respectively. DNA and RNA were extracted and processed as described in the legend to Fig. 1 and in Materials and Methods.



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DISCUSSION

In this study we have evaluated the influence of the adeno-viral E4 region on the strength and persistence of transgeneexpression from the CMV promoter in vitro and in vivo. Usinga series of isogenic E1/E3 deletion vectors with specific mod-ifications in the E4 region, we confirm and extend earlierobservations (1, 10) that the viral E4 region can regulate in cisand in trans the strength and stability of transgene expressionfrom the CMV promoter, and we show that the E4 ORF3function plays a pivotal role in this genetic regulation. In ad-dition, the biological activity of E4 ORF3 was found to bemodulated, as additional viral E4 functions, such as ORF4 orORF6 and ORF6/7 and/or tissue-specific cellular factors, arerequired to sustain transgene expression in vivo.

This study used vectors carrying the hCFTR gene under thecontrol of the CMV promoter as a model system. Given thehuman origin of the transgene, the in vivo evaluation of thevectors was performed in parallel in immunodeficient SCIDmice and immunocompetent C3H and C57BL/6 mice in orderto determine the influence of the host immunological responseagainst hCFTR on the persistence of transgene expression. Inthe presence of the wild-type E4 region, extended transgenepersistence could be observed in SCID and C57BL/6 mice butnot in C3H or CBA mice. This stable expression of the trans-gene in C57BL/6 mice was correlated with a relatively stablepersistence of the viral genome in the transduced cells and withan absence of a detectable anti-hCFTR cellular immune re-sponse. In contrast, the viral DNA copy number, and hencehCFTR expression, rapidly declined to undetectable levels inCBA and C3H mice, in correlation with the induction of acellular anti-hCFTR immune response. Since all immunocom-petent mice developed similar antiviral immune responses,these data support our previous results indicating that the hostimmune response directed against the transgene product playsa predominant role in controlling the in vivo persistence of thetransduced cells (14, 40, 43). These results also indicate thatC57BL/6 mice may constitute attractive hosts for the in vivoevaluation of vectors for cystic fibrosis gene therapy, since theyseem to be fully immunologically responsive to the adenoviralantigens but less responsive to the hCFTR protein. This ob-

servation is consistent with previous reports showing extendedexpression of transgenes encoding secreted human proteins (2,43, 45, 57, 59), correlated with an impaired antibody responseagainst the secreted human proteins, in C57BL/6 mice but notin other strains of mice. In contrast, Scaria et al. (53) recentlydescribed extended CMV-hCFTR expression in the lungs ofimmunocompetent mice, including C3H and BALB/c mice.These authors suggested that under the conditions described,the hCFTR protein was virtually nonimmunogenic, and theycorrelated the extended transgene expression with a lack of ananti-hCFTR immune response. Whether the differences in vec-tor backbones contribute to the differing results observed re-mains to be clarified.

In contrast to the stable transgene expression obtained withthe E1/E3 deletion vector containing the wild-type E4 region,CMV-driven transgene expression was shut off with the E1/E3/E4 deletion vector regardless of the immune status of theanimals. However, the patterns of transgene expression in theliver and the lung differed from each other. No hCFTR mRNAcould be detected at any time in the livers of mice treatedintravenously with the E1/E4 deletion vector, while stronghCFTR expression was observed at day 3 postinjection in thelungs of mice treated intratracheally with the same vector. Inthe latter case, however, transgene expression was unstableand declined to undetectable levels 2 weeks later. Interestingly,the E1/E3/E4 deletion vector was reproducibly less toxic andinflammatory in the livers of immunocompetent mice than theAdE1° vector (15). Together, these results suggest direct in-volvement of the E4 gene products in gene expression andvector toxicity.

To further investigate the mechanism(s) of E4-mediatedregulation of gene expression from the CMV promoter, a se-ries of isogenic CMV-hCFTR expression vectors differing onlyin the E4 region, and containing individual E4 ORFs or com-binations of E4 ORFs, was generated. These vectors weretested for transgene expression in vitro and in SCID mice inthe absence of a specific host immune response. The majorfinding from this study is that the E4 ORF3 gene product isabsolutely required for long-term transgene expression fromthe CMV promoter. However, depending on the target tissue,

FIG. 10. E4 ORF3 is sufficient for the persistence of CMV-CFTR expression in the livers of SCID mice. Vectors with wild-type (wt) E4 sequences or with specificmodifications in the E4 region were administered by intravenous injection to SCID mice at a dose of 1.5 3 109 IU/animal, with five animals per time point. Animalswere sacrificed on the indicated days. The persistence of viral DNA (A) and of hCFTR expression (B) in the liver were determined by Southern and Northern blotanalysis, respectively. DNA and RNA were extracted and processed as described in the legend to Fig. 1 and in Materials and Methods.



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transgene expression is regulated either by ORF3 alone or byORF3 together with specific additional E4 gene products.Thus, in the liver, ORF3 was required and sufficient for boththe establishment and the long-term maintenance of transgeneexpression. However, the establishment of strong initial CMV-driven hCFTR expression in the liver was delayed by a few dayscompared to that with the vector carrying the wild-type E4region. In the lung, no E4 gene product was required for theestablishment of transgene expression, but ORF3 was neces-sary, although not sufficient, for the long-term maintenance oftransgene expression. Sustained transgene expression in thelung required the cooperation of ORF3 with either ORF4 orORF6 and ORF6/7.

The phenotypic description of E4 functions has evolvedthrough the genetic and molecular analysis of viral mutantswith modifications only in E4, and little is known about theinfluence of E4 gene products on the regulation of heterolo-gous transcription units. The viral E4 region encodes severalregulatory functions which seem to act in a pleiotropic fashionin transcription, accumulation, splicing, and transport of earlyand late mRNAs, in DNA replication, and in virus assembly(reviewed in references 38 and 55). For example, it was shownthat the E4 ORF3 and ORF6 proteins increase the productionof the viral late proteins by facilitating the cytoplasmic accu-mulation of the relevant mRNAs at a posttranscriptional level(38, 55). The redundant functions of ORF3 and ORF6 innuclear RNA stabilization may be linked directly to RNAsplicing. Both proteins have been shown to affect viral RNAsplicing patterns (48, 49). ORF3 promotes exon inclusion inboth viral major late-gene-derived transcripts and nonviraltranscripts, while ORF6 promotes exon exclusion. Both ORF3and ORF6 also play redundant roles in viral DNA replication.However, the ORF3 function is dispensable, whereas theORF6 function is absolutely required for viral growth, at leastin vitro (8, 9, 33). Given the complex effects of the ORF3 andORF6 proteins on viral gene expression and viral replication, abetter characterization of the interactions of these proteinswith the host cell components is critical for understanding theinfluence of these proteins on heterologous gene expression. Inthis context, it has recently been shown that ORF3 alone, evenin the absence of viral infection, can directly affect the distri-bution of a group of essential transcription/replication factorsin the nucleus (11, 23, 24).

Whether this function of ORF3 is related to the requirementfor the ORF3 protein for establishment and maintenance oftransgene expression, reported here, is currently unclear. How-ever, the results from our in vitro nuclear run-on assays un-equivocally demonstrate that the mechanism(s) underlying theregulation of transgene expression from the CMV promoter inthe presence of either ORF3 alone, ORF3, ORF4, andORF3/4, or ORF3, ORF6, and ORF6/7 includes transcrip-tional activation of the CMV promoter by the E4 functions.These results are entirely consistent with the observation thatadministration of a vector without a transgene but retainingthe E4 functions to animals previously injected with an E1/E3/E4 deletion vector carrying a CMV-driven expression cas-sette results in reactivation in trans of the CMV promoter inthe E1/E3/E4 deletion vector (see Results) (10).

Our studies also show that E4 ORF3 must cooperate eitherwith ORF4 or with ORF6 and ORF6/7 to maintain persistenttransgene expression in the lung, while ORF3 is sufficient forstable CMV-driven transgene expression in the liver, despite adelay in the kinetics of hCFTR expression. It has been re-ported that ORF4 regulates protein phosphorylation in in-fected cells by binding to the cellular protein phosphatase 2A(36, 46). This interaction results in the selective hypophosphor-

ylation of several cellular and viral proteins, including E1A andthe c-Fos component of the AP1 transcription factor. It is notclear whether this function of ORF4 is important, togetherwith ORF3, for the persistence of heterologous transgene ex-pression in the lung. Alternatively, regulation of transgeneexpression in the presence of ORF3 and ORF4 could also bedue to an ORF3/4 function. Such a putative ORF3/4 protein ispredicted to exist based on analysis of the viral mRNA gener-ated in Ad2-infected HeLa cells (60), but its existence has notyet been experimentally demonstrated. The molecular mecha-nisms of regulation of transgene expression by ORF3 togetherwith ORF6 and ORF6/7 also remain unclear. The ORF6/7product has been shown to bind to the E2F cellular transcrip-tion factor and to modulate its activity (34). While there are noapparent E2F binding sites in the CMV promoter, the ORF6/7or ORF6 product, in concert with the ORF3 protein, couldrecruit critical cellular transcription factors to modulate CMVpromoter activity. Interestingly, several additional, as yet un-identified, cellular proteins have recently been shown to inter-act with the E4 ORF6 and ORF6/7 proteins (5).

Despite the poor characterization of the mechanisms bywhich the viral E4-encoded proteins regulate the activity ofheterologous promoter sequences, this study clearly shows thatthe presence of E4 ORF3 is necessary and, at least in the liver,sufficient for stable in vivo transgene expression. This impliesthat the ORF3 protein can act alone or can cooperate witheither ORF4 or ORF6 and ORF6/7, and with specific cellularfactors, to control the expression of candidate therapeuticgenes. Moreover, our studies demonstrate that the E4 productsare involved in regulation of the CMV promoter at the tran-scriptional level. Further studies are required to more preciselyinvestigate the mechanisms of the E4-dependent activation oftranscription and to determine whether other heterologouspromoters are similarly regulated by the E4-encoded proteins.This hypothesis is supported by our observation that geneexpression from the RSV promoter was also modulated by theE4 region. Whether cellular promoters are also sensitive to E4is currently under investigation.

Altogether, these studies reemphasize the notion that thearchitecture of the vector backbone and the choice of thetransgene transcription unit constitute important parametersdictating the persistence of transgene expression. Interestingly,the viral E4 region appears to be directly involved in thehepatotoxicity and inflammation profile of the vector as well.Deletion of the E4 region, in addition to E1, markedly de-creased the toxicity of the vector and the host inflammationresponse. Low liver toxicity was also observed for AdE1°E4°vectors retaining either ORF3 alone or ORF3, ORF4, andORF3/4. In contrast, vectors retaining ORF6 and ORF6/7,with or without ORF3, or ORF4 alone displayed high livertoxicity (15). Thus, E1/E3/E4 deletion vectors retaining a func-tional E4 ORF3 or a combination of ORF3 and ORF4 com-bine the desirable feature of long-term transgene expressionwith low toxicity and low inflammatory responses. Such vectorsmight be useful for further liver- or lung-directed gene therapyapplications.

ACKNOWLEDGMENTS

We are grateful to R. Rooke and M. Courtney for their criticalcomments and suggestions on the manuscript. We thank K. Schughartfor the coordination of the animal studies.

This work was supported in part by the Association Francaise contrela Mucoviscidose (AFLM) and the Association Francaise contre lesMyopathies.



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ADDENDUM IN PROOF

While the article was under review, we noted that transgeneexpression in the liver was very weak in the absence of E1 andE4 as early as 6 h postinfection and declined to undetectablelevels by 3 days postinfection. In contrast, expression in thepresence of E4 was strong and stable during these early timepoints.

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