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Further Reduction in Adenovirus Vector-Mediated Liver Transduction without Largely Affecting Transgene Expression in Target Organ by Exploiting MicroRNA-Mediated Regulation and the Cre-loxP Recombination System David Bennett,† Fuminori Sakurai,*,† Kahori Shimizu,† Hayato Matsui,† Kyoko Tomita,† Takayuki Suzuki,‡ Kazufumi Katayama,† Kenji Kawabata,‡,§ and Hiroyuki Mizuguchi*,†,‡,∥ †

Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Osaka, Japan § Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan ∥ Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan ‡

ABSTRACT: In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3′-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3′-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen. KEYWORDS: adenovirus vector, Cre recombinase-loxP system, microRNA, targeted expression

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promoters, including a glial fibrillary acidic protein (GFAP) gene promoter,4 a synthetic muscle-specific promoter,5 and the human alpha1-antitrypsin promoter (hAAT) containing two copies of the hepatic control region of the apolipoprotein E (ApoE) enhancer,6 have been used as tissue-specific promoters in previous studies. Another approach to modifying transgene expression has been to utilize tissue-specific endogenous microRNA (miRNA) expression to detarget transgene expression from specific tissues.7,8 MicroRNAs (miRNAs) are endogenously expressed ∼22-nucleotide noncoding RNAs involved in post-transcriptional gene regulation.9 Through binding to complementary sites in the 3′ untranslated region (UTR), miRNAs suppress gene expression by preventing the translation and/or hastening

INTRODUCTION Adenovirus (Ad) vectors possess a number of desirable properties that have made them a commonly used tool in gene therapy and basic research. For instance, Ad vectors are capable of efficiently transducing a wide range of cells and lack the mutagenic potential of genome-incorporating viruses such as retroviruses. In order to achieve safe and effective gene therapy using Ad vectors, Ad vectors capable of tissue-specific transgene expression would be desirable. However, systemically administered conventional Ad vectors mediate remarkably high transduction in the liver, making the targeted transgene expression in tissues other than the liver challenging. There have been several different approaches for development of targeted Ad vectors. One approach is altering the tropism of Ad vectors by genetically modifying the capsid proteins, including incorporation of a foreign peptide or protein into the fiber knob, hexon, and protein IX.1−3 In another approach, tissue-specific promoters are used to target transgene expression to specific tissues. Various types of tissue-specific © 2012 American Chemical Society

Received: Revised: Accepted: Published: 3452

May 2, 2012 September 23, 2012 November 2, 2012 November 5, 2012 dx.doi.org/10.1021/mp300248u | Mol. Pharmaceutics 2012, 9, 3452−3463

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Table 1. Ad Vectors Used in This Studya

the degradation of target mRNA.9,10 While some miRNAs are expressed ubiquitously, many miRNAs show different expression patterns in different cell subsets, and at different stages in development. The typical procedure for placing a transgene under the regulation of miRNA is to incorporate target sequences complementary to miRNA into the 3′-UTR of the transgene. By incorporating sequences complementary to miRNAs that are expressed exclusively in specific cell subsets or at certain developmental stages, this approach has been used to detarget transgene expression from cell types as diverse as hepatocytes,11−14 muscle cells,15 and neurons.16 Furthermore, including target sequences complementary to multiple miRNAs can detarget expression of a single gene from multiple cell types.12,15,17 However, even with miRNA-medited suppression, some levels of transgene expression still remain, especially when the transgene is highly expressed. Insertion of miR-122a target sequences into the 3′-UTR of the transgene loaded in the Ad vector suppressed the transgene expression in the liver by about 100-fold; however, significant levels of Ad vector-mediated transgene expression were still found in the liver.11 In order to further suppress the Ad vector-mediated transgene expression in the liver, another approach should be included in addition to the miRNA-regulated transgene expression system. However, such an approach should minimize disturbing transgene expression in target organs. In this study, we developed a novel Ad vector system utilizing both miRNA-mediated regulation and the Cre recombinase (Cre)-loxP system to further reduce hepatic transduction without significantly suppressing transgene expression in target organs. In this system, one Ad vector carries a transgene expression cassette containing loxP sites and miR-122a target sequences. Cre expression cassette is included in a separate Ad vector. In addition, target sequences against miR-142-3p, which is highly expressed in the spleen,18 the target organ of this study, were inserted into the 3′-UTR of Cre gene in order to reduce Cre expression in the spleen. The spleen is a suitable target for immunotherapy because the spleen possesses specialized compartments where immune cells gather and work. Efficient transgene expression in the spleen induces superior vaccine effects against transgene products.19 We found that our Ad vector system is capable of extremely efficient suppression of transgene expression in the liver while largely maintaining transgene expression in the spleen.

vector name

promoter

Ad-L2

CMV

Ad-LacZ Ad-AHA-L

CMV AHA

Ad-SV40-hAlb-L Ad-L-lox

SV40hAlb CMV

Ad-L-lox-122aT

CMV

Ad-LacZ-lox122aT Ad-L-RLb

CMV CMV

Ad-L-142-3pT-RLb

CMV

Ad-CMV-Cre

CMV

Ad-CMV-Cre-1423pT Ad-AHA-Cre

CMV

Ad-AHA-Cre-1423pT Ad-AHA-FLP-1423pT Ad-null

AHA

AHA

AHA 

transgene

miRNA target sequence

loxP

firefly luciferase β-galactosidase firefly luciferase firefly luciferase firefly luciferase firefly luciferase β-galactosidase





 

 







○

miR-122a

○

miR-122a

○

firefly luciferase firefly luciferase Cre recombinase Cre recombinase Cre recombinase Cre recombinase FLP recombinase 





miR-142-3p







miR-142-3p







miR-142-3p



miR-142-3p







a

The transgene expression cassettes described in the table were inserted into the El-deleted region of the Ad vector genome. , Ad vector does not contain the corresponding sequences, including miRNA target sequence and loxP sequence. ○, Ad vector possesses the corresponding sequences. bA renilla luciferase expression cassette was included into the E3-deleted region of the Ad vector genome.

promoter composed of apolipoprotein E enhancer, the hepatocyte control region, and human α1-antitrypsin promoter (AHA promoter)6 were kindly provided by Dr. Mark A. Kay (Stanford University, CA). Human albumin promoter fused with SV40 enhancer (SV40-hAlb promoter) was derived from pDRIVE-SV40-hAlb (Invivogen, San Diego, CA). LoxP and FRT sequences are previously reported.23,24 For insertion of 4 copies of the sequences perfectly complementary to miRNAs into the 3′-UTR of the transgene, synthetic oligonucleotides were inserted into the plasmids. Detailed information about the construction of the plasmids and oligonucleotides for miRNA target sequences is available from the authors on request. For construction of the Ad vector plasmids, the shuttle plasmids described above were digested by I-CeuI/PI-SceI and ligated with I-CeuI/PI-SceI-digested pAdHM4,20 producing the Ad vector plasmids containing the transgene expression cassette in the E1-deleted region. To generate the Ad vectors, the Ad vector plasmids were digested with PacI to release the recombinant viral genome. Linearized DNA was transfected into 293 cells using SuperFect transfection reagent (Qiagen, Hilden, Germany). All Ad vectors were propagated in 293 cells, purified by two rounds of cesium chloride-gradient ultracentrifugation, dialyzed, and stored at −80 °C. The virus particle (VP) titer was determined by a spectrophotometrical method.25 In Vitro Transduction into Cultured Cell Lines. HeLa cells were seeded in a 96-well plate at 3.5 × 103 cells/well. On the following day, the cells were transduced with Ad-AHA-Cre

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EXPERIMENTAL SECTION Cells and Mice. Huh-7 cells and 293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) and antibiotics. HeLa cells were cultured in minimal essential medium (MEM) supplemented with 10% FCS and antibiotics. All cells were cultured at 37 °C in humidified air containing 5% CO2. Female C57/BL6 mice aged 5−7 weeks were obtained from Nippon SLC (Hamamatsu, Japan). Ad Vectors. The Ad vectors used in this study, which were constructed by an improved in vitro ligation method,20−22 are shown in Table 1. The firefly luciferase gene, renilla luciferase gene, and β-galactosidase (LacZ) gene were derived from pGL3-control (Promega, Madison, WI), pGL4.70 (Promega), and pCMVβ (Clontech, Mountain View, CA), respectively. The CMV promoter (human cytomegalovirus immediate-early 1 gene promoter) and BGHpA (polyadenylation signal sequence from the bovine growth hormone gene) were derived from pHMCMV6.20 Cre gene and a synthetic liver-specific 3453

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Figure 1. A two-Ad vector system for further suppression of hepatic transduction without largely affecting transduction in the spleen by utilizing both the miRNA-regulated transgene expression system and the Cre-loxP system. (A) Schematic diagram of the Cre- and reporter gene-expressing Ad vectors used in this study. The Cre gene contains target sequences to spleen-specific miR-142-3p in the 3′-UTR in order to detarget Cre expression from the spleen. The reporter gene is flanked by loxP sequences and contains target sequences to liver-specific miR-122a in the 3′-UTR in order to detarget reporter gene expression from the liver. Cre- and reporter gene-expressing Ad vectors are intravenously coinjected. (B) In the liver, Cre is expressed and excises the loxP-flanked reporter gene, suppressing transcription of the reporter gene. Binding of miR-122a to target sites suppresses residual reporter gene mRNA. In the spleen, transcription of the Cre gene occurs but miR-142-3p binds to target sites in the Cre gene mRNA, suppressing Cre expression. Transcription and translation of reporter gene are unimpeded, and reporter gene is expressed at high levels. CMV, cytomegalovirus immediate early promoter; AHA, the liver-specific apolipoprotein E enhancer-hepatocyte control region-human α1antitrypsin promoter; pA, bovine growth hormone polyadenylation signal; Luc, firefly luciferase; LacZ, β-galactosidase; Cre, Cre recombinase; loxP, 34-bp loxP sequence.

as described above. Total DNA, including Ad vector genome, was recovered from the cells using DNAzol (Invitrogen, Carlsbad, CA) after a total 72 h incubation for HeLa cells and total 48 h incubation for Huh-7 cells. PCR analysis was performed in 25 μL of the reaction mixture containing total DNA, 0.625 unit Ex taq DNA polymerase (TAKARA BIO INC, Otsu, Japan), 2 mM MgCl2, and 0.2 mM dNTP. The sequences of the primers targeting the BGH pA and the pIX gene in the Ad vector genome are as follows: the BGHpA-F, 5′TAGAAGGCACAGTCGAGG-3′; pIX-R, 5′-TCACCTTTACCACGTCCTGG-3′. The following parameters were used: 10 s

or Ad-CMV-Cre at 150 VP/cell for 1.5 h. Following a 48 h incubation, the cells were transduced with Ad-L2 or Ad-L-lox at 150 VP/cell. Following another 48 h incubation, luciferase expression was determined using a PicaGene LT2.0 luciferase assay system (Toyo Inki, Tokyo, Japan). Huh-7 cells were seeded in a 96-well plate at 1 × 104 cells/well. On the following day, the cells were transduced with 150 VP/cell of Ad-AHACre or Ad-CMV-Cre and 150 VP/cell of Ad-L2 or Ad-Luc-lox. The cells were cultured for 72 h and luciferase expression determined as for HeLa cells. PCR Analysis for Cre-loxP System-Mediated Excision of Luciferase Gene. HeLa and Huh-7 cells were transduced 3454

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at 98 °C and 180 s at 68 °C for 35 cycles. The PCR products were electrophoresed in 0.7% agarose gels. In Vivo Transduction by Ad Vectors Containing Various Promoters. Promoter activity for transgene expression in the organs was examined by intravenous administration of Ad vectors containing either CMV, AHA, or SV40-hAlb promoter-driven firefly luciferase expression cassette at a dose of 1 × 1010 VP/mouse. Two days after injection, firefly luciferase production in the organs was determined as previously described.26 In Vivo Transduction by Ad Vectors Containing miR142-3p Target Sequences. The in vivo suppressive effects of the insertion of miR-142-3p target sequences in the transgene expression cassette were examined by intravenous administration of Ad vectors containing miR-142-3p target sequences in the firefly luciferase expression cassette (Ad-L-142-3pT-RL) at the dose of 1 × 1010 VP/mouse. Ad-L-142-3pT-RL was constructed similarly as Ad-L-122aT, which has been previously constructed.11 As a control Ad vector, Ad-L-RL, which was identical to Ad-L described in the previous study,11 was used. Two days after injection, firefly and renilla luciferase expression levels in the organs were determined. In Vivo Transduction Following Coadministration of Cre-Expressing Ad Vector and Reporter Gene-Expressing Ad Vector. Mice were intravenously injected with 1 × 1010 VP of luciferase-expressing Ad vector (Ad-L-lox or Ad-L-lox122aT) mixed with 1 × 1010 VP of Ad-CMV-Cre, Ad-CMVCre-142-3pT, or Ad-AHA-FLP-142-3pT. Ad-AHA-FLP-1423pT, which expresses FLP recombinase and shows no loxPspecific recombinase activity, was used as a control Ad vector. Three days following injection, luciferase expression levels in the organs were determined. In order to analyze the optimal ratio of Cre-expressing Ad vector and reporter gene-expressing Ad vector, mice were injected via the tail vein with 1 × 1010 VP of Ad-L-lox-122aT and 0, 0.25, 1, or 4 × 1010 VP of Ad-CMV-Cre-142-3pT, along with Ad-null, which possesses no transgene expression cassette and was used as a control to balance the total dose of the Ad vectors to 5 × 1010 VP/mouse. Three days following injection, luciferase expression levels in the organs were determined. In Vivo Transduction Following LacZ-Expressing Ad Vector Administration. Mice were injected via the tail vein with 1 × 1010 VP of Ad-LacZ or Ad-LacZ-lox-122aT mixed with 1 × 1010 VP of Ad-AHA-FLP-142-3pT or Ad-CMV-Cre-1423pT. Three days following injection, the liver and spleens were harvested from the mice and embedded in Tissue-Tek OCT (Miles, Elkhart, IN). 10 μm cryostat sections were prepared, dried for 1 h at room temperature, and then fixed in 0.5% glutaraldehyde-phosphate buffered saline (PBS) for 5 min. Following wash with PBS, X-gal staining was performed as previously described.27 Sections were examined using a microscope. Statistical Analysis. Statistical significance was determined by the t test. Statistical significance was defined as P < 0.05.

complementary to liver-specific miR-122a into the 3′-UTR. In order to further reduce the Ad vector-transduction in the liver, the reporter gene was flanked by loxP sites, and the reporter gene-expressing Ad vector and the Cre-expressing Ad vector were coadministered. In the liver, the reporter gene expression should be suppressed by miR-122a. Furthermore, the reporter gene expression should be suppressed by loxP site-specific excision of the reporter gene from the Ad vector genome by Cre expressed by the other Ad vector. In order to maintain the transgene expression in the spleen as much as possible, Ad vector-mediated Cre expression in the spleen was suppressed by insertion of sequences perfectly complementary to spleenspecific miR-142-3p. Comparison of Promoter Activity for Expression of Cre in the Organs. First, in order to select promoters for Ad vector-mediated Cre expression, we compared the in vivo activities of the ubiquitous CMV promoter and two liverspecific promoters, the SV40-hAlb promoter and AHA promoter, in Ad vectors following systemic administration in mice (Figure 2). In the system described in Figure 1, Cre

Figure 2. Luciferase production in the organs following intravenous administration of Ad vectors possessing various promoters. Mice were intravenously injected with 1 × 1010 VP of the Ad vectors. Two days following injection, luciferase expression in the organs was measured using a luciferase assay system. Data are the mean ± SE (n = 4) and are representative of two independent experiments.

should ideally be highly expressed in a liver-specific pattern. The CMV promoter mediated luciferase production in all of the organs measured, with production in the liver much higher than in other organs (approximately 104-, 103-, 105-, and 105fold higher production in the liver than in the spleen, heart, kidney, and lung, respectively). Substantial levels of luciferase production were found in the organs other than the liver. The AHA and SV40-hAlb promoters exhibited luciferase expression in a liver-specific manner. The organs other than the liver expressed background levels of luciferase by the AHA and SV40-hAlb promoter, although a very low but nonetheless detectable level of luciferase expression was found in the heart for the AHA promoter. In the liver, of the two liver-specific promoters, the AHA promoter showed 31-fold higher activity than the SV40-hAlb promoter. The CMV promoter activity was 32-fold higher than that of the AHA promoter. Based on these data, the AHA and CMV promoters were used for expressing Cre in the subsequent experiments. In Vitro Transgene Expression Profiles Following the Combinational Transduction of Cre-Expressing Ad Vector and miRNA-Regulated Reporter Gene-Expressing Ad Vector. Next, to examine whether CMV promoter- or AHA promoter-driven Cre expression efficiently drives the CreloxP system, cells were cotransduced with luciferase-expressing Ad vectors (Ad-L2 or Ad-L-lox) and Cre-expressing Ad vectors

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RESULTS The aim of this study was to develop a system for further reduction in Ad vector-mediated liver transduction without largely affecting transduction in the spleen. For this purpose, we combined the miRNA-regulated transgene expression system and the Cre-loxP system (Figure 1). The reporter gene, which was inserted into the E1-deleted region of the Ad vector genome, was modified by the insertion of sequences perfectly 3455

dx.doi.org/10.1021/mp300248u | Mol. Pharmaceutics 2012, 9, 3452−3463

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Figure 3. In vitro luciferase expression following transduction with luciferase-expressing Ad vector and Cre-expressing Ad vector. HeLa and Huh-7 cells were transduced with the indicated luciferase- and Cre-expressing Ad vectors. Cre expression was driven by the AHA (A) or CMV promoters (B). Luciferase production was determined 48 h (HeLa cells) or 72 h (Huh-7 cells) after transduction. Data are shown as % luciferase expression relative to that in the cells transduced with Ad-L2 and Ad-CMV-Cre or Ad-AHA-Cre. Data are the mean ± SD (n = 3). *: P < 0.05.

Figure 4. PCR analysis for Cre-mediated excision of luciferase gene from the Ad vector genome following in vitro transduction. Schematic diagram of the PCR analysis for the Cre-mediated excision is shown in (A). HeLa (B) and Huh-7 cells (C) were transduced as described in Figure 3. Total DNA, including the Ad vector genome, was recovered from the cells, and the PCR analysis was performed using primers targeting the BGHpA and the pIX gene in the Ad vector genome. M, 1kb ladder; arrow 1, unexcised forms of the luciferase-expressing Ad vector genomes; arrow 2, Creexpressing Ad vector genomes; arrow 3, excised form of the luciferase-expressing Ad vector genome. Abbreviations in the figure are the same as those in Figure 1.

(Ad-AHA-Cre or Ad-CMV-Cre). Cotransduction with AdAHA-Cre, the Ad vectors expressing Cre by the AHA

promoter, efficiently suppressed luciferase expression from Ad-L-lox by 13-fold in the hepatocellular carcinoma cell line 3456

dx.doi.org/10.1021/mp300248u | Mol. Pharmaceutics 2012, 9, 3452−3463

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Huh-7 cells, but did not significantly reduce the luciferase expression in HeLa cells (Figure 3A), presumably due to hepatocyte-specific expression of Cre by the AHA promoter. Expression of Cre by Ad-CMV-Cre largely reduced luciferase expression by Ad-L-lox, by 17- and 208-fold in HeLa and Huh7 cells, respectively (Figure 3B). The reduction level by AdCMV-Cre in the luciferase expression was larger than that by Ad-AHA-Cre due to the stronger promoter activity of the CMV promoter than AHA promoter as shown in Figure 2. We confirmed that, even with the presence of loxP sites in the luciferase expression cassette, insertion of miR-122a target sequences into the luciferase expression cassette efficiently suppressed luciferase expression in Huh-7 cells, but not HeLa cells (data not shown), as shown in our previous study.11 To further examine whether the luciferase gene in the Ad vector genome was successfully excised by Cre, PCR analysis was performed (Figure 4). In both HeLa and Huh-7 cells, bands for the excised form (Arrow 3) were not found following cotransduction with Ad-L2 and Ad-CMV-Cre or Ad-AHA-Cre because there were no loxP sequences in the genome of Ad-L2. On the other hand, the luciferase gene was efficiently excised in both HeLa and Huh-7 cells cotransduced with Ad-L-lox and Ad-CMV-Cre. Co-transduction with Ad-L-lox and Ad-AHACre also resulted in efficient excision of luciferase gene in Huh7 cells; however, the band intensity of the unexcised form (Arrow 1) was slightly higher for Ad-L-lox and Ad-AHA-Cre than that for Ad-L-lox and Ad-CMV-Cre in Huh-7 cells due to higher levels of Cre expression by the CMV promoter than the AHA promoter. The clear band corresponding to the excised form was not found in HeLa cells following cotransduction with Ad-L-lox and Ad-AHA-Cre because the liver-specific AHA promoter did not efficiently transcribe the Cre gene in HeLa cells. It was unclear why the band for Ad-CMV-Cre (Arrow 2) was not apparent in HeLa cells cotransduced with Ad-CMVCre and Ad-L-lox. These results indicated that the Cre-loxP system in the Ad vectors functioned successfully following transduction. Suppression of Transgene Expression in the Spleen by Insertion of miR-142-3p Target Sequences. In order to examine the suppressive activity of miR-142-3p target sequences on Ad vector-mediated transgene expression in the spleen, Ad-L-142-3pT-RL, in which miR-142-3p target sequences were inserted into the 3′-UTR of the firefly luciferase gene, was administered to the mice. The renilla luciferase gene was also incorporated into the E3-deleted region of the vector in order to normalize the firefly luciferase gene expression in Ad-L-142-3pT-RL. Intravenous administration of Ad-L-142-3pT-RL resulted in 4-fold reduction in the firefly luciferase expression in the spleen, compared with that by AdL-RL, which does not possess miR-142-3p target sequences in the 3′-UTR of firefly luciferase gene (Figure 5). There was no significant difference in the firefly luciferase expression in the liver between Ad-L-142-3pT-RL and Ad-L-RL. These results indicated that insertion of miR-142-3p target sequences in the 3′-UTR of transgene suppressed the transgene expression in a spleen-specific manner. In Vivo Transduction Profiles Following the Combinational Transduction of Ad Vectors Employing miRNARegulated Transgene Expression System and the CreloxP System. Next, in order to test whether combinational transduction of Ad vectors employing miRNA-regulated transgene expression system and the Cre-loxP system further reduce reporter gene expression in the liver without largely

Figure 5. In vivo luciferase expression following intravenous administration of an Ad vector containing miR-142-3p target sequences in the firefly luciferase expression cassette. Mice were intravenously injected with 1 × 1010 VP of Ad-L-RL or Ad-L-142-3pTRL. Two days following injection, luciferase expression in the organs was measured using a luciferase assay system. Data are the mean ± SE (n = 4). *: P < 0.05.

affecting reporter gene expression in the spleen, the luciferaseexpressing Ad vectors and Cre-expressing Ad vectors were coadministered to mice. As shown in our previous study,11 mice injected with Ad-L-lox-122aT showed 161-fold decreased luciferase expression in the liver, compared to mice injected with Ad-L-lox (Figure 6A). In the other organs including the spleen, there was a reduction of less than 3-fold in the luciferase expression for Ad-L-lox-122aT, compared with Ad-L-lox (Figure 6B−E) (note that the y-axis in Figure 6A is logarithmic scale, however those in Figure 6B−E are normal scale). This reduction in Ad-L-lox-122aT-mediated transduction in organs other than the liver was probably due to nonspecific inhibition by insertion of miR-122a target sequences. Levels of miR-122a expression in organs other than the liver were almost undetectable (data not shown). Coadministration of AdAHA-Cre or Ad-AHA-Cre-142-3pT failed to significantly reduce the luciferase expression in the organs, including the liver, compared with coadministration of Ad-AHA-FLP-1423pT, indicating that Cre expression in the organs by Ad-AHACre and Ad-AHA-Cre-142-3pT was too low to induce the recombination. 55-fold reduction in the luciferase expression in the liver was found for coadministration of Ad-CMV-Cre, compared with coadministration of Ad-AHA-FLP-142-3pT; however, the luciferase expression in the spleen was also significantly reduced by Ad-CMV-Cre, probably due to the efficient Cre expression in the spleen by Ad-CMV-Cre. On the other hand, insertion of the miR-142-3p target sequences restored the luciferase expression in the spleen; similar levels of luciferase expression were found for Ad-L-lox-122aT/Ad-CMVCre-142-3pT and Ad-L-lox-122aT/Ad-AHA-FLP-142-3pT. Coadministration of Ad-CMV-Cre-142-3pT resulted in 24-fold reduction in the luciferase expression in the liver, compared with coadministration of Ad-AHA-FLP-142-3pT. These results indicated that the coadministration of Ad vectors expressing Cre by the CMV promoter, but not AHA promoter, reduced the luciferase expression by Ad-L-lox-122aT to lower levels than that by Ad-L-lox-122aT alone. Furthermore, suppression of the luciferase expression in the spleen by Ad-CMV-Cre was 3457

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Figure 6. Luciferase expression in the organs following coadministration of luciferase-expressing and Cre-expressing Ad vectors. Mice were coadministered with 1 × 1010 VP each of luciferase-expressing Ad vector (Ad-L-lox or Ad-L-lox-122aT) and Cre-expressing Ad vector (Ad-AHA-Cre, Ad-AHA-Cre-142-3pT, Ad-CMV-Cre, or Ad-CMV-Cre-142-3pT) or Ad-AHA-FLP-142-3pT, which has no loxP-specific recombinase activity and was used as a control. Three days after injection, luciferase expression in the organs was determined. Data are shown for the liver (A), spleen (B), heart (C), kidney (D), and lung (E). The data are the mean ± SE (n = 6) and are representative of two independent experiments. *: P < 0.05 compared with Ad-L-lox-122aT/Ad-AHA-FLP-142-3pT group. N.D., not detected.

prevented by including the spleen-specific miR-142-3p target sequences into the 3′-UTR of the Cre gene. Taken together, approximately 3800-fold reduction in the transduction in the liver was achieved by coadministration of Ad-L-lox-122aT and Ad-CMV-Cre-142-3pT, compared with Ad-L2 and Ad-AHAFLP-142-3pT. On the other hand, coadministration of Ad-Llox-122aT and Ad-CMV-Cre-142-3pT resulted in only 3.7-fold reduction in the transduction efficiency in the spleen. In this experiment, total 2 × 1010 VP of Ad vectors was intravenously administered to mice. Significant liver toxicity was not found following administration (data not shown). Effects of Dosage Ratio of the Reporter GeneExpressing Ad Vector and Cre-Expressing Ad Vectors on Transduction Profiles. To examine how altering the ratio of Cre-expressing Ad vector to luciferase-expressing Ad vector affected the in vivo reporter gene expression patterns, mice were administered with various ratios of Ad-L-lox-122aT and AdCMV-Cre-142-3pT. Luciferase expression in the liver was

reduced proportionally to the amount of Ad-CMV-Cre-1423pT (Figure 7A). When the ratio of Ad-L-lox-122aT to AdCMV-Cre-142-3pT was 4, 123-fold reduction in the luciferase expression in the liver was found, compared with expression by Ad-L-lox-122aT alone. The luciferase expression in the spleen was also reduced proportionally to Ad-CMV-Cre-142-3pT (Figure 7B), however, these reductions were relatively small compared to the reduction in the luciferase expression in the liver (note that the y-axis in Figure 7A is logarithmic scale, however that in Figure 7B is normal scale). These data indicated that increasing the amount of coadministered Creexpressing Ad vector largely decreased the absolute reporter gene expression in the liver. Localization of the Reporter Gene-Expressing Cells in the Organs Following the Combinational Transduction. In order to visually examine the localization of cells expressing the reporter gene in the organs following the combinational transduction, a LacZ gene was included in our Ad vector system 3458

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Figure 7. Effect of dosage ratios of the luciferase-expressing Ad vector and Cre-expressing Ad vector on the luciferase expression profiles in the liver and spleen. Mice were intravenously coadministered with Ad-L-lox-122aT and Ad-CMV-Cre-142-3pT at the indicated amounts, with Ad-null used as a control Ad vector to balance the total Ad vector amount of all injections to 5 × 1010 VP/mouse. Three days after administration, luciferase production in the liver (A) and spleen (B) was determined. Data are the mean ± SE (n = 6) and are representative of two independent experiments. N.D., not detected.

unwanted transgene expression in the liver for a range of applications. Increasing the ratio of Cre-expressing Ad vector to luciferaseexpressing Ad vector in our system further decreased the luciferase expression in the liver; however, the spleen also showed a small decrease in the luciferase expression (Figure 7). Applications using this system should select a ratio of a Creexpressing Ad vector and a transgene-expressing Ad vector based on both the relative and absolute expression levels of transgene desired in the liver and spleen. The spleen was targeted by our Ad vector system in this study because its crucial role in immune responses makes it an attractive target for immunotherapy. Target sequences for spleen-specific miR-142-3p were inserted into the Cre expression cassette in order to maintain the reporter gene expression in the spleen. Transduced cells in the spleen were predominantly located in the marginal zone, which is an area dense in immune cells, particularly B cells, macrophages, and DCs.28 Previous studies, including ours, demonstrated that the marginal zone of the spleen was efficiently transduced by Ad vectors.29−31 Furthermore, our group has shown that intravenously injected Ad vector gives high transgene expression in spleen DCs,32 suggesting that the Ad vector system of this study is transducing DCs of the marginal zone. High transgene expression specifically in DCs following intravenous injection of an Ad vector could be useful for vaccination, in which genes encoding antigens and immunostimulatory proteins are delivered to DCs. Efficient transgene expression in the spleen results in superior immune responses against transgene products.19 Spleen-specific transgene expression is an important goal for reducing the toxicity of Ad vector-based vaccines, as transduced nontarget cells that present the antigens on their surface are recognized and destroyed by the immune system. Following coadministration of Ad-L-lox-122aT and AdCMV-Cre-142-3pT, transduction in the liver was dramatically reduced by 3777-fold while transduction efficiency in the spleen was reduced only by 3.7-fold, compared with those following coadministration of Ad-L-Lox and Ad-AHA-FLP-142-3pT. Nevertheless, despite this dramatic detargeting of Ad vector transduction from the liver, the absolute transgene expression levels in the liver were still comparable to that in the spleen

instead of the luciferase gene. PBS-injected mice showed no Xgal staining activity (Figure 8A,E). Insertion of the miR-122a target sequences significantly suppressed the LacZ expression in the liver; however, there was still LacZ activity in a high proportion of cells in the liver (Figure 8C). In the spleen, LacZ expression was observed in the marginal zone (Figure 8F), an area containing dendritic cells (DCs), marginal zone B cells, and macrophages.28 Interestingly, the coadministration of AdCMV-Cre-142-3pT almost completely suppressed LacZ expression in the liver (Figure 8D) but LacZ expression in the marginal zone was largely unaffected (Figure 8H). These results also indicated that Ad vector-mediated combinational transduction with the miRNA-regulated transgene expression system and Cre-loxP system significantly reduced the hepatic transduction without reducing the transgene expression in the spleen.

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DISCUSSION In this study, we developed a new approach for further reducing reporter gene expression in the liver without largely affecting the reporter gene expression in the spleen by using both the miRNA-regulated transgene expression system and the CreloxP system. Our system gives extremely efficient suppression of transgene expression in the liver and shows substantial transgene expression in the marginal zone of the spleen. Although 4 copies of miR-122a target sequences inserted into the 3′-UTR of transgene reduced the luciferase expression in the liver by 161-fold (Figure 6), a high proportion of hepatocytes still showed high LacZ expression (Figure 8), indicating that the insertion of miR-122a target sequences alone was insufficient in suppressing the Ad vector-mediated transgene expression in the liver. The coadministration of Ad vectors possessing a CMV promoter-driven Cre expression cassette reduced the transgene expression in the liver by an additional 24-fold for Ad-CMV-Cre-142-3pT and 55-fold for Ad-CMV-Cre, compared with the coadministration of the control Ad vector. This equated to a reduction of 3777-fold for Ad-CMV-Cre-142-3pT and 8839-fold for Ad-CMV-Cre over the coadministration of Ad-L-lox and Ad-AHA-FLPe-142-3pT. This system may therefore be a valuable tool to prevent 3459

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Figure 8. LacZ expression in the liver and spleen following intravenous coadministration of the LacZ-expressing Ad vector and Cre-expressing Ad vector. (A−D) liver, (E−H) spleen. Mice were intravenously coadministered with either PBS (A, E), Ad-LacZ (B, F), Ad-LacZ-lox-122aT and AdAHA-FLP-142-3pT (C, G), or Ad-LacZ-lox-122aT and Ad-CMV-Cre-142-3pT (D, H) at the dose of 1 × 1010 VP of each Ad vector. Three days after coadministration, tissue sections were prepared from the liver and spleen. Sections were X-gal-stained overnight, and then counterstained with nuclear fast red. Scale bar at the bottom indicates 100 μm. The results are representative of three independent experiments.

following coadministration of Ad-L-lox-122aT and Ad-CMVCre-142-3pT. However, as described above, transgene ex-

pression in the spleen was concentrated to the marginal zone, in which immune cells, including DCs, are localized. We 3460

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sulfate.37−40 Furthermore, several studies have demonstrated that blood coagulation factor X (FX) directly binds to the hexon, leading to efficient transduction in the hepatocytes through the interaction between FX and heparan sulfate on the cell surface.41−44 Modification of the Ad capsid proteins described above has reduced hepatic transduction. In particular, Alba et al. demonstrated that a hexon-modified Ad vector mediated higher transduction efficiencies in the spleen than the liver following intravenous administration.31 Combined use of capsid-modified Ad vector, the Cre-loxP system, and the miRNA-regulated trangene expression system would result in more efficient spleen-specific transgene expression. In conclusion, we have developed a two-Ad vector system that uses the miRNA-regulated transgene expression system and the Cre-loxP system. This system is extremely efficient in suppressing transgene expression in the liver while maintaining substantial transgene expression in the marginal zone of the spleen. This system might have important potential for Ad vector-mediated targeted transduction and for the manipulation of spleen immune cells in vivo.

consider that coadministration of Ad-L-lox-122aT and AdCMV-Cre-142-3pT mediated a substantial and sufficient level of transgene expression in the marginal zone of the spleen to exhibit therapeutic value. We confirmed that intravenous administration of 1 × 1010 VP of Ad-LacZ into mice resulted in induction of significant levels of LacZ-specific CD8+ cells in the spleen (data not shown). In this study, sequences perfectly complementary to miR142-3p, which is highly expressed in the spleen, were incorporated into the 3′-UTR of the Cre gene to suppress Cre expression and thereby maintain the reporter gene expression in the spleen. It is possible that this Ad vector system could be adapted to use different tissue-specific or tumor-specific miRNA sequences in order to detarget Ad vector-mediated Cre expression to give specific transgene expression in other tissues or in tumors. Cre-expressing Ad vectors using the AHA promoter showed no significant reduction in the luciferase expression in the liver, despite the AHA promoter exhibiting the transcriptional activity specifically in the liver. On the other hand, Creexpression driven by the CMV promoter, which gave 32-fold higher transcriptional activity in the liver than the AHA promoter, resulted in efficient loxP site-specific recombination in the liver. These results suggested that although the AHA promoter drove Cre expression in the liver, the Cre expression levels were probably insufficient to mediate the recombination in the luciferase expression cassette inserted in the Ad vector genome. The data of Figure 2 demonstrated that transgene expression mediated by the AHA promoter was 32-fold lower than the CMV promoter in the liver. A stronger liver-specific promoter may be capable of attaining Cre levels sufficient to mediate the recombination. Miao et al. demonstrated that the AHA promoter was strengthened by approximately 10-fold by including the 1.4 kb truncated factor IX intron A.6 Sequestration of Ad vectors from the bloodstream by liver Kupffer cells can be a major obstacle for efficient transduction following systemic administration. Polyinosinic acid-mediated blockade of Ad vector uptake by liver Kupffer cells or depletion of liver Kupffer cells by clodronate liposomes enhances Ad vector-mediated transduction in the liver.33−35 Sequestration of Ad vectors by liver Kupffer cells could also be a problem for the Ad vector-delivered Cre-loxP system used in this study because the Cre-expressing Ad vector and reporter gene-expressing Ad vector must both transduce each hepatocyte in order to suppress the transgene expression in the liver. Tao et al. demonstrated in mice that although intravenous administration of 1 × 1011 VP/mouse of an Ad vector achieved highly efficient transduction, administration of the same vector at 1−3 × 1010 VP/mouse gave barely detectable levels of transgene expression.36 This suggests that the majority of the injected dose was sequestered by the reticuloendothelial system (RES), including liver Kupffer cells, in the dose range of 1−3 × 1010 VP/mouse.36 However, contrary to these findings, in our present study almost all hepatocytes were transduced following intravenous administration of 1 × 1010 VP of Ad-LacZ (Figure 8), suggesting that both Cre-expressing Ad vector and reporter gene-expressing Ad vector cotransduced the hepatocyctes even at the dose of 1 × 1010 VP/mouse of each Ad vector. Ad vectors efficiently transduce hepatocytes following intravenous administration via several pathways, including interaction between fiber knob and coxsackievirus-adenovirus receptor (CAR) on the hepatocytes, RGD (Arg-Gly-Asp) motif in the penton base and αv-integrins, and fiber shaft and heparin

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AUTHOR INFORMATION

Corresponding Author

*Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan. Fax: +81-6-68798186. F.S.: tel, +81-6-6879-8188; e-mail, [email protected]. ac.jp. H.M.: tel, +81-6-6879-8185; e-mail, mizuguch@phs. osaka-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Takako Ichinose (National Institute of Biomedical Innovation, Osaka, Japan), Sayuri Okamoto, Masuo Kondoh, and Kiyohito Yagi (Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan) for their help. This work was supported by a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and a grant from the Takeda Science Foundation.

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