Germline Incorporation of a Replication-Defective Adenoviral Vector in Mice Does Not Alter Immune Responses to Adenoviral Vectors
Authors: Fernando D. Camargo, Dorothy A. Huey-Louie, Aloke V. Finn, André B. Sassani, Aaron E. Cozen, Hideaki Moriwaki, Darren B. Schneider, Ramtin Agah, David A. Dichek
Journal: Molecular Therapy (2000)

The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a “null” (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P < 0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens. The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a “null” (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P < 0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens. IntroductionThe utility of adenoviral vectors for in vivo gene transfer is limited by immune responses to adenoviral proteins. These responses limit the duration of transgene expression after an initial vector exposure, interfere with readministration of the vector, and cause inflammation in target tissues (1Yang Y. Li Q. Ertl H.C.J. Wilson J.M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.J. Virol. 1995; 69: 2004-2015Crossref PubMed Scopus (1000) Google Scholar, 2Dai Y. et al.Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allows for long-term expression.Proc. Natl. Acad. Sci. USA. 1995; 92: 1401-1405Crossref PubMed Scopus (581) Google Scholar, 3Newman K.D. et al.Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.J. Clin. Invest. 1995; 96: 2955-2965Crossref PubMed Scopus (270) Google Scholar). The immune response to adenoviral vectors can be mitigated by deleting and eliminating viral open reading frames (4Engelhardt J.F. Ye X. Doranz B. Wilson J.M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver.Proc. Natl. Acad. Sci. USA. 1994; 91: 6196-6200Crossref PubMed Scopus (579) Google Scholar, 5Kochanek S. et al.A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and β-galactosidase.Proc. Natl. Acad. Sci. USA. 1996; 93: 5731-5736Crossref PubMed Scopus (498) Google Scholar) or by immune suppression of the recipient (6Fang B. et al.Gene therapy for hemophilia B: Host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.Hum. Gene Ther. 1995; 6: 1039-1044Crossref PubMed Scopus (117) Google Scholar). However, both of these approaches have shortcomings. Even the complete elimination of viral open reading frames may not prevent generation of an immune response to viral proteins in the initial viral inoculum. Moreover, immune suppression may alter the cellular and organ physiology in a recipient animal (7Hojo M. et al.Cyclosporine induces cancer progression by a cell-autonomous mechanism.Nature. 1999; 397: 530-534Crossref PubMed Scopus (962) Google Scholar, 8Sudhir K. et al.Cyclosporine impairs release of endothelium-derived relaxing factors in epicardial and resistance coronary arteries.Circulation. 1994; 90: 3018-3023Crossref PubMed Google Scholar). As a result, phenotypes obtained in gene transfer studies performed in immunosuppressed animals may be difficult to attribute solely to effects of a transferred gene.We sought to establish an experimental setting in which immune-competent animals could be exposed to adenoviral vectors without provocation of an immune response. In this setting, the long-term consequences of transgene expression could be determined in the absence of confounding effects of immune responses or immune suppression. In addition, the absence of neutralizing antibody formation could permit vectors to be readministered as needed. To this end, we sought to develop lines of immune-competent mice in which the immune response to adenoviral antigens was selectively absent because the mice were transgenic for, and therefore tolerant of adenovirus.In addition to the possibility of generating mice that are tolerant to adenoviral vectors, production of adenovirus-transgenic mice could reveal the consequences of germline integration and transmission of an adenoviral vector genome. Adenovirus can integrate into mammalian chromosomal DNA (9Harui A. Suzuki S. Kochanek S. Mitani K. Frequency and stability of chromosomal integration of adenovirus vectors.J. Virol. 1999; 73: 6141-6146Crossref PubMed Google Scholar), and several protocols for vector administration result in dissemination of adenovirus to gonadal tissues (10Barr E. et al.Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus.Gene Ther. 1994; 1: 51-58PubMed Google Scholar, 11Kass-Eisler A. et al.The impact of developmental stage, route of administration and the immune system on adenovirus-mediated gene transfer.Gene Ther. 1994; 1: 395-402PubMed Google Scholar, 12Ye X. et al.Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse.Hum. Gene Ther. 1998; 9: 2135-2142Crossref PubMed Scopus (61) Google Scholar). Although germline transmission of adenoviral vectors appears infrequent (12Ye X. et al.Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse.Hum. Gene Ther. 1998; 9: 2135-2142Crossref PubMed Scopus (61) Google Scholar), it is not impossible and therefore must be considered among the potential side effects of human gene therapy. Germline integration of an adenoviral vector might cause specific abnormalities related to production of viral proteins. Alternatively, if tolerance to adenoviral proteins is present in adenovirus-transgenic offspring, they might be susceptible to overwhelming adenoviral infection.In this paper, we describe the generation and propagation of several lines of adenovirus-transgenic mice and report on the nature of their immune responses after a postnatal exposure to adenoviral vectors.Materials and MethodsGeneration and identification of adenovirus-transgenic mice. We considered two approaches to generate adenovirus-transgenic mice: injection of zygotes with an intact E1-deleted adenoviral genome or, alternatively, injection of multiple individual expression cassettes in which each of the several adenoviral transcription units was driven by a thymus-specific promoter. For several reasons we chose the former strategy. First, immunologic tolerance exists to both endogenous as well as transgenic proteins that are expressed only in extrathymic (i.e., peripheral) tissues (13Nossal G.J.V. Negative selection of lymphocytes.Cell. 1994; 76: 229-239Abstract Full Text PDF PubMed Scopus (721) Google Scholar). Second, expression of antigens in the thymus does not assure tolerance (14Salmon A.-M. et al.An acetylcholine receptor α subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.J. Clin. Invest. 1998; 101: 2340-2350Crossref PubMed Scopus (44) Google Scholar). Third, expression of adenoviral antigens from endogenous adenoviral promoters in E1-deleted vectors is well documented (4Engelhardt J.F. Ye X. Doranz B. Wilson J.M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver.Proc. Natl. Acad. Sci. USA. 1994; 91: 6196-6200Crossref PubMed Scopus (579) Google Scholar). Expression from these promoters occurs via either E1A-independent transcription or by complementation of E1A functions by cellular factors (15Dooley T.P. Miranda M. Jones N.C. DePamphilis M.L. Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos.Development. 1989; 107: 945-956PubMed Google Scholar, 16Reichel R. Kovesdi I. Nevins J.R. Developmental control of a promoter-specific factor that is also regulated by the E1A gene product.Cell. 1987; 48: 501-506Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Therefore, one would expect expression from an integrated E1A-deleted adenoviral genome, especially during development (15Dooley T.P. Miranda M. Jones N.C. DePamphilis M.L. Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos.Development. 1989; 107: 945-956PubMed Google Scholar). Fourth, there are numerous primary transcripts from the adenoviral genome and these primary transcripts are spliced in a complex pattern (17Shenk T. Adenoviridae: The viruses and their replication.in: Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd ed. Lippincott–Raven, Philadelphia1996: 2111-2148Google Scholar). Breaking up the adenoviral genome into separate expression cassettes would likely interfere with expression of at least some of the adenoviral proteins. Finally, there is precedent for achieving tolerance to viral antigens such as hepatitis B core antigen or herpes simplex virus glycoprotein D by expressing these antigens in peripheral tissues of transgenic mice (18Abramczuk J.W. et al.Transgenic mouse lines that are immunologically tolerant and nontolerant to herpes simplex virus glycoprotein D.J. Virol. 1991; 65: 6124-6128PubMed Google Scholar, 19Milich D.R. et al.Extrathymic expression of the intracellular hepatitis B core antigen results in T cell tolerance in transgenic mice.J. Immunol. 1994; 152: 455-466PubMed Google Scholar).Transgenic mice were generated by injection of zygotes of FVB/N mice, according to standard protocols (20Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1994Google Scholar). Zygotes were injected with DNA (2 ng/μl) purified from the AdNull adenoviral vector (Fig. 1A). This vector (21Kang S.-M. et al.Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction.Nat. Med. 1997; 3: 738-743Crossref PubMed Scopus (428) Google Scholar) contains a full-length adenovirus 5 genome with deletions in the E1 (0.9 to 9.8 map units) and E3 regions (77.5 to 86.2 map units). Transgenic mice were identified by Southern blotting of HindIII-digested tail DNA. Blots were hybridized with genomic DNA of AdNull, which was labeled with 32P by the random-priming method. FVB/N mice were used because of the ease with which transgenesis can be accomplished in this strain (22Taketo M. et al.FVB/N: An inbred mouse strain preferable for transgenic analyses.Proc. Natl. Acad. Sci. USA. 1991; 88: 2065-2069Crossref PubMed Scopus (492) Google Scholar), the utility of this line for similar studies (18Abramczuk J.W. et al.Transgenic mouse lines that are immunologically tolerant and nontolerant to herpes simplex virus glycoprotein D.J. Virol. 1991; 65: 6124-6128PubMed Google Scholar, 23Leonard J.M. et al.Development of disease and virus recovery in transgenic mice containing HIV proviral DNA.Science. 1988; 242: 1665-1670Crossref PubMed Scopus (138) Google Scholar), and a lack of recognized immunological peculiarities in FVB/N mice, as reported in an informal survey of colleagues. Mice were housed in a barrier facility under pathogen-free conditions. All animal protocols were approved by the Committee on Animal Research of the University of California, San Francisco.Northern analysis. Organs were harvested from adult transgenic and nontransgenic mice and total RNA was extracted with Trizol (Life Technologies, Grand Island, NY). RNA (10 μg) from each organ was separated by gel electrophoresis and analyzed by Northern blotting, as described (24Dong G. Schulick A.H. DeYoung M.B. Dichek D.A. Identification of a cis-acting sequence in the human plasminogen activator inhibitor type-1 gene that mediates transforming growth factor-β1 responsiveness in endothelium in vivo.J. Biol. Chem. 1996; 271: 29969-29977Crossref PubMed Scopus (39) Google Scholar). To enable detection of transcripts from all areas of the integrated adenoviral genome, Northern blots were probed with 32P-labeled DNA of AdNull, the same probe used for Southern blots (see above). As a positive control, 2 μg of RNA from adenovirus-infected 293 cells was electrophoresed, blotted, and probed in parallel. As expected, hybridization of the adenovirus DNA to RNA from adenovirus-infected 293 cells revealed numerous abundant transcripts (not shown).Preparation and infusion of adenoviral vectors. Postnatal mice were injected with either of two recombinant adenoviral vectors: AdhAAT and AdCMVNull. AdhAAT, which expresses a human alpha-1 antitrypsin (hAAT) cDNA from a cytomegalovirus (CMV) promoter, was provided by Dr. M. Kay (Stanford University). AdCMVNull is an E1, E3-deleted vector that is identical to AdNull, except that a 1.1-kb HinPI fragment of plasmid pcDNA3 (Invitrogen) containing the CMV promoter and bovine growth hormone polyadenylation signal (but without an intervening transgene) is present at the site of the E1 deletion (Fig. 1B). Concentrated vector stocks were prepared, stored, and characterized as described (25Lee S.W. et al.In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries.Circ. Res. 1993; 73: 797-807Crossref PubMed Scopus (195) Google Scholar). All stocks were free of E1A-containing virus as shown by a PCR-based assay that can detect one E1A-containing genome per 1 × 106 vector genomes.Viral vectors (3 × 108 PFU in 100 μl) were infused via the jugular vein. AdhAAT was injected only in nontransgenic mice. AdCMVNull was injected in both AdNull-transgenic mice and in nontransgenic littermate controls. AdCMVNull was detected by digestion of liver DNA with BamHI and hybridization of Southern blots to a 32P-labeled BglII–EcoRI fragment of plasmid pCI (Invitrogen), which contains the CMV promoter but no adenoviral sequences. To aid in quantitation of AdCMVNull DNA, each Southern blot contained a standard curve of electrophoresed and blotted CMV promoter DNA corresponding to 0.2 to 20 copies per cell in the experimental lanes.AdCMVNull DNA was also detected by performing real-time “TaqMan” quantitative PCR on template liver DNA (26Gibson U.E.M. Heid C.A. Williams P.M. A novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1765) Google Scholar). Liver DNA (500 ng) was combined with primers (5′-CAT CTA CGT ATT AGT CAT CGC TAT TAC CA-3’ and 5′-TGG AAA TCC CCG TGA GTC A-3′) that amplify a 94-bp sequence in the CMV promoter. Amplified product was detected with a probe to an intervening sequence: 5′-ACC GCT ATC CAC GCC CAT TGA TGT-3′. The amplification and detection protocol was similar to that described elsewhere (27Becker K. Pan D. Whitley C.B. Real-time quantitative polymerase chain reaction to assess gene transfer.Hum. Gene Ther. 1999; 10: 2559-2566Crossref PubMed Scopus (38) Google Scholar). Samples were assayed in duplicate; all duplicate amplifications yielded signals differing by <5%. To permit quantitation of AdCMVNull DNA in experimental samples, a standard curve was included in which amplifications were performed in wells spiked with 102 to 106 copies of pcDNA3. These amounts of plasmid DNA correspond to a range of 0.0022 to 22 copies of CMV promoter sequence per diploid cell in the experimental samples. Amplifications in the standard wells generated fluorescence signals that were linear over this range (r2 = 0.99). Nearly all (30 of 31; 97%) of the experimental signals were within the standard curve. No CMV sequences were detected in liver DNA from a mouse not injected with AdCMVNull.Alpha-1 antitrypsin expression. hAAT protein and antibodies directed against hAAT were measured by ELISA performed on mouse serum, as described (28Schowalter D.B. et al.Constitutive expression of murine CTLA4Ig from a recombinant adenovirus vector results in prolonged transgene expression.Gene Ther. 1997; 4: 853-860Crossref PubMed Scopus (41) Google Scholar). Samples were assayed in triplicate.Antibodies to adenovirus. Serum antibodies to adenovirus were measured by ELISA at 2, 4, 6, 8, and 12 weeks after injection of adenovirus. Briefly, 96-well plates were coated by incubating them with 100 μl of vector stock containing 1 × 109 adenoviral particles. The wells were rinsed and blocked with 1.5% bovine serum albumin, 0.1% Tween 20 in phosphate-buffered saline. Diluted mouse serum (100 μl) was added to each well for 90 min at 4°C. Bound antibody was detected with alkaline phosphatase-conjugated anti-mouse IgG (Sigma No. A2429), IgG1 (Zymed No. 61-0122), or IgG2a (Zymed No. 61-0222) and p-nitrophenyl phosphate substrate (Sigma). Each sample was assayed in triplicate. Serum from naive mice was used as a negative control. Serum from adenovirus-injected mice reliably gave signals above control values.Splenocyte proliferation assay. Splenocytes were harvested at 4 and 8 weeks after injection of AdCMVNull and assayed for proliferation in response to adenoviral antigens, as described (29Coligan J.E. et al.Current Protocols in Immunology. Wiley, New York1991Google Scholar). Briefly, spleens were disrupted and filtered through Nylon mesh, and splenocytes were plated in 96-well plates at 5 × 105 cells/well. UV-inactivated adenovirus was added to achieve virion:splenocyte ratios of 2:1, 10:1, and 50:1. Only the 50:1 ratio reliably stimulated proliferation of splenocytes from AdCMVNull-injected mice. Higher amounts of virus were toxic. As a positive control, concanavalin A (5 ng/ml; Sigma) or phytohemagglutinin (10 ng/ml; Sigma) was added to the medium instead of adenovirus. As a negative control, splenocytes were cultured in medium only. On day 3 or 4, cells were pulsed overnight with [3H]thymidine (0.5 μCi/well; Amersham). Labeled DNA was collected with a cell harvester (Tomtec, Hamden, CT) and counted with a scintillation counter. The means of six wells per experimental condition were used to calculate the proliferative index (i.e., the ratio of [3H]thymidine incorporation in virus-stimulated wells to [3H]thymidine incorporation in negative control wells). Because proliferative indices were low (≤2) in splenocyte assays performed with the AdCMV-Null-injected FVB/N mice, we repeated the proliferation assay twice with splenocytes from BALB/c mice (Jackson Laboratories, Bar Harbor, ME).Statistics. Birth ratios were compared by χ2 test and litter sizes by unpaired t test. Groups of transgenic and nontransgenic mice were, with one exception, compared by unpaired t tests. The splenocyte proliferation data (in which not all data were normally distributed) were analyzed by Kruskal–Wallis ANOVA with Dunn's method for correcting for multiple pairwise comparisons (30Zar J.H. In Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ1984: 306-327Google Scholar).ResultsAdenovirus-Transgenic MiceOf 31 pups delivered by mothers implanted with zygotes injected with AdNull DNA, 7 (22%) were transgenic for AdNull. One of the founders did not transmit the transgene. A second founder died and was found postmortem to have an ovarian teratoma. The remaining 5 founders were fertile, transmitted the transgene, and showed no gross abnormalities. Southern analysis of tail tip DNA from these lines revealed 2 to 50 copies of intact AdNull per haploid genome (Fig. 2). As expected, the presence of a 1.4-kb junction fragment indicated integration of head-to-tail concatemers of AdNull DNA (see Fig. 1A).FIG. 2Southern analysis of tail-tip DNA from AdNull-transgenic mice. DNA (15 μg) from one mouse in each of the five transgenic lines (1Yang Y. Li Q. Ertl H.C.J. Wilson J.M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.J. Virol. 1995; 69: 2004-2015Crossref PubMed Scopus (1000) Google Scholar, 2Dai Y. et al.Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allows for long-term expression.Proc. Natl. Acad. Sci. USA. 1995; 92: 1401-1405Crossref PubMed Scopus (581) Google Scholar, 5Kochanek S. et al.A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and β-galactosidase.Proc. Natl. Acad. Sci. USA. 1996; 93: 5731-5736Crossref PubMed Scopus (498) Google Scholar, 6Fang B. et al.Gene therapy for hemophilia B: Host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.Hum. Gene Ther. 1995; 6: 1039-1044Crossref PubMed Scopus (117) Google Scholar, 7Hojo M. et al.Cyclosporine induces cancer progression by a cell-autonomous mechanism.Nature. 1999; 397: 530-534Crossref PubMed Scopus (962) Google Scholar) was digested with HindIII, electrophoresed, blotted, and probed with 32P-labeled AdNull DNA. HindIII-digested AdNull DNA was also analyzed in parallel (left lane). The size of the fragments of AdNull (in kb) is indicated. All expected fragments are present (see Fig. 1), although the 0.075-kb fragment is too small to detect. In the mouse DNA, the 1.0- and 0.4-kb bands are absent, and a new 1.4-kb band is present, as expected with integrated, concatamerized DNA (see Fig. 1A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)All five lines were bred to generate animals for experimentation. A total of 36 litters was generated (5 to 9 litters/line; Table 1). Genotyping revealed significant underrepresentation of transgenic pups: of 255 total pups, only 108 (42%) were transgenic (P < 0.02). Notably, only three of the five lines contributed to the skewed birth rate. In each of these lines, 36–39% of pups were transgenic, compared to 50% in the two other lines. These data suggested that the transgene conferred embryonic lethality with incomplete penetrance. If this were true, litters of the three lines with skewed birth rates might be smaller than litters of the other two lines. Indeed, litters (n = 24) of the skewed lines included 6.4 ± 2.3 pups versus 8.5 ± 1.6 pups in litters (n = 12) of the nonskewed lines (P = 0.007).TABLE 1Birth Data and Genotypes in Five Lines of AdNull-Transgenic MiceLineTotal littersTotal pupsTransgenic pupsPercentage transgenesis1949193928582136576030506746173775422150Total36255108*P < 0.02 Versus expected frequency of 50%.42*P < 0.02 Versus expected frequency of 50%.* P < 0.02 Versus expected frequency of 50%. Open table in a new tab Expression of Adenoviral Transcripts in Adenovirus-Transgenic MiceTo determine whether adenoviral transcripts were expressed, we analyzed RNA from organs of three transgenic and two nontransgenic adult mice. Because we sought to detect any of numerous (≥30) transcripts initiated throughout the adenoviral genome (17Shenk T. Adenoviridae: The viruses and their replication.in: Fields B.N. Knipe D.M. Howley P.M. Fields Virology. 3rd ed. Lippincott–Raven, Philadelphia1996: 2111-2148Google Scholar), we probed Northern blots with full-length adenoviral genomic DNA. In two nontransgenic and two transgenic mice, we detected no adenoviral transcripts in brain, heart, kidney, liver, lung, muscle, and spleen. However, in one transgenic mouse (data not shown), we detected adenoviral transcripts in the spleen (4.9 kb) and the lung (0.6 kb). These data confirm a low level of postnatal expression from the integrated adenoviral genome. We cannot exclude the possibility that other viral transcripts are present at other time points, in other tissues, or at levels too low to detect by Northern analysis. For several reasons (see below) we did not perform a more comprehensive analysis of expression and relied instead on functional assays to investigate whether transgenesis for adenovirus resulted in immunologic tolerance and vector persistence.Adenovirus-Mediated hAAT ExpressionWe tested whether hAAT would serve as a useful reporter for following the persistence of adenoviral vectors infused in AdNull-transgenic FVB/N mice. The antigenicity of hAAT in mice is strain-dependent, and it is a useful reporter gene only in strains (e.g., C57BL/6) in which it is not antigenic (31Barr D. et al.Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: Comparisons between immunocompetent and immunodeficient inbred strains.Gene Ther. 1995; 2: 151-155PubMed Google Scholar). In three of four FVB/N mice injected intravenously with AdhAAT, expression of hAAT was stable over 9 weeks and then slowly declined (Fig. 3A and data not shown). In one of the mice, however, hAAT expression declined by over 99% within 4 weeks (Fig. 3A); this mouse also had high serum levels of hAAT-reactive antibodies (Fig. 3B). Thus, hAAT is not a useful reporter for investigating the persistence of adenoviral vectors in FVB/N mice because these mice can mount an immune response to hAAT, leading to loss of transgene expression and, potentially, loss of vector DNA. This immune response could obscure an underlying tolerance to adenoviral proteins expressed by the AdhAAT backbone.FIG. 3Expression of hAAT and generation of anti-hAAT antibodies after intravenous injection of AdhAAT. Four nontransgenic FVB/N mice were injected with AdhAAT, and their serum was assayed for hAAT protein and antibodies at weekly intervals. Each mouse is represented by a separate symbol. Mouse 2 (triangles) rapidly lost expression and simultaneously developed a high titer of antibodies to hAAT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Persistence of Vector DNABecause AdhAAT was not a suitable vector, we used a null virus (which does not contain a potentially immunogenic reporter gene) to investigate persistence of vector DNA in AdNull-transgenic mice. AdNull-transgenic mice already have AdNull DNA in every cell (2 to 50 copies; Fig. 2); therefore, we infused AdCMVNull, which contains CMV promoter sequences (but no transgene) in addition to adenoviral DNA (Fig. 1B). We detected AdCMVNull DNA in livers of AdNull-transgenic mice by Southern analysis and real-time PCR. Both techniques were designed to detect the CMV promoter sequences present in AdCMVNull but absent in AdNull.AdNull-transgenic mice and littermate controls from all five lines (at least eight transgenic and eight nontransgenic mice in each line) were injected intravenously with 3 × 108 pfu of AdCMVNull. Southern analysis of liver DNA was performed on all lines at 4 weeks, three lines at 8 weeks, and one line at 10 to 12 weeks(Table 2). CMV promoter sequences (0.2 to 2 copies per diploid cell) were detected in liver DNA from nearly all AdCMVNull-injected mice. There was no difference in the amount of vector DNA in transgenic versus nontransgenic mice (Fig. 4A). Consistent with the hAAT data (Fig. 3)