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Background/aim: Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome., Materials and Methods: We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined., Results: The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers., Conclusion: The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for self-replication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd., Materials and Methods: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds., Results: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds., Conclusion: These results provide important clues for the development of safe and efficient OAds., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4 , leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.

Background: Arming of an oncolytic adenovirus (OAd) by inserting expression cassettes of therapeutic transgenes into the OAd genome is a promising approach to enhance the therapeutic effects of an OAd. Ideally, this approach would simultaneously promote the replication of an OAd in tumor cells and transgene product-mediated antitumor effects by expressing therapeutic transgenes. We previously demonstrated that knockdown of cullin 4A (CUL4A), which is an E3 ubiquitin ligase, significantly promoted adenovirus replication by increasing the c-JUN protein level. In addition, previous studies reported that CUL4A was highly expressed in various types of tumor, and was involved in tumor growth and metastasis., Materials and Methods: In this study, we developed a novel OAd expressing a short-hairpin RNA (shRNA) against CUL4A (OAd-shCUL4A)., Results: OAd-shCUL4 mediated higher levels of cytotoxic effects on various types of human tumor cell than a conventional OAd. Higher levels of OAd genome copy numbers were found in the tumor cells for OAd-shCUL4A, compared with a conventional OAd., Conclusion: OAd-shCUL4A showed efficient antitumor effects by both enhancing OAd replication and inhibiting tumor cell growth., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection. IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from VA-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on host-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad., (Copyright © 2019 American Society for Microbiology.)

Pre-existing anti-adenovirus (Ad) neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using Ad vectors and oncolytic Ads; however, it has not been fully elucidated which Ad capsid protein-specific antibodies are involved in AdNAb-mediated inhibition of Ad infection in vivo. In this study, mice possessing antibodies specific for each Ad capsid protein were prepared by intramuscular electroporation of each Ad capsid protein-expressing plasmid. Ad vector-mediated hepatic transduction was efficiently inhibited by more than 100-fold in mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid. An Ad vector pre-coated with FX before administration mediated more than 100-fold lower transduction efficiencies in the liver of warfarinized mice immunized with a fiber protein-expressing plasmid or a penton base-expressing plasmid, compared with those in the liver of warfarinized non-immunized mice. These data suggest that anti-fiber protein and anti-penton base antibodies bind to an Ad vector even though FX has already bound to the hexon, and inhibit Ad vector-mediated transduction. This study provides important clues for the development of a novel Ad vector that can circumvent inhibition with AdNAbs.

Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP) 50 titer as a measure of viral quality. The GFP 50 titer (GFP 50 /mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP 50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP 50 /mL and tissue culture infectious dose (TCID 50 /mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP 50 /mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.

Cell-penetrating peptides (CPPs) have been highly anticipated as an efficient delivery system due to their ability to cross biological membranes and transport various cargoes into cells. In the present study, we have identified adenovirus-derived CPPs using various capsid-mutant adenovirus (Ad) vectors. First, we examined the endocytosis-inducing ability of these vectors. A fiber-shaft substituted Ad vector, Ad type 5 vector with the fiber shaft domain replaced by that derived from Ad type 35, induced the highest fluorescein isothiocyanate (FITC)-dextran uptake into a human liver cell line, HepG2 cells. In contrast, the FITC-dextran uptake in HepG2 cells was not significantly different between coxsackievirus and adenovirus receptor (CAR)-binding-ablated Ad vector, integrin-binding-ablated Ad vector or conventional Ad vector. Next, we produced a recombinant Ad type 35 shaft protein using the Escherichia coli recombinant system. The recombinant Ad type 35 shaft protein retained the ability for FITC-dextran uptake and efficient gene delivery by plasmid transfection reagent. Furthermore, we identified 26 C-terminal amino acids in the Ad type 35 shaft protein as the cell membrane binding domain. The 26 amino-acid peptides also have the potential to be internalized into cultured cells. The internalization ability of the peptide was dependent on degree and was inhibited by an actin polymerization inhibitor (Latrunculin B) and by a lipid raft formation inhibitor (methyl-β-cyclodextrin). The results of the present study indicate that Ad type 35-derived peptides induce endocytosis in cultured cells and have the ability to cross biological membranes. This report is the first paper to identify Ad-derived CPPs.

Oncolytic viruses have been receiving much attention as potential agents for cancer treatment. Among the various types of oncolytic viruses, the telomerase-specific replication-competent adenovirus (TRAD), which carries the tumor-specific promoter-driven E1 gene expression cassette, exhibits efficient antitumor effects. The development of a novel TRAD that shows higher replication efficiency and antitumor activity would be highly beneficial for safer and more efficient cancer therapy. We recently demonstrated that the endoribonuclease Dicer significantly inhibits the replication of wild-type adenovirus (Ad) via the processing of viral-associated (VA)-RNAs, which are Ad-encoded small noncoding RNAs, and that the knockdown of Dicer leads to enhanced VA-RNA expression and Ad replication after infection with wild-type Ad. Based on these findings, we herein developed a novel TRAD expressing short-hairpin RNA against Dicer (shDicer; TRAD-shDicer). After infection, TRAD-shDicer efficiently induced the knockdown of Dicer. TRAD-shDicer showed significantly higher replication efficiency and tumor cell lysis activity compared with the conventional TRAD in tumor cells. The Dicer expression levels and viabilities of normal cells were not altered by infection with TRAD-shDicer. These results indicate that TRAD-shDicer is a potent antitumor reagent by virtue of its enhanced oncolytic activity. Mol Cancer Ther; 16(1); 251-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Adenovirus vectors (Adv) elicit innate immune responses via several pattern-recognition receptors. Although it has been suggested that various Adv-induced mechanisms play important roles in the induction of innate immunity in vitro, the impacts of these mechanisms in vivo remain unclear. Viral nucleic acids elicit innate immune responses through the recognition of cytosolic nucleic acid sensors and transduce intracellular signals to TANK-binding kinase (TBK) 1. In this study, to determine the impacts of viral nucleic acids on innate immune responses in vivo, we administered transgene-expressing Adv to Tbk1-deficient mice. The systemic Adv administration failed to induce type I interferons (type I IFNs) in the spleen, but not the liver, of Tbk1-deficient mice, resulting in the increase of transgene-expressing cells in the spleen, but not the liver. Moreover, Adv failed to induce type I IFNs in the bone-marrow-derived dendritic cells, but not the mouse embryonic fibroblasts, from Tbk1-deficient mice in vitro. These results support the idea that Adv elicit innate immunity in immune cells and non-immune cells in a TBK1-dependent and TBK1-independent manner, respectively., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of life via retro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

Few of the vaccines in current use can induce antigen- (Ag-) specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m.) vaccination of an adenovirus vector (Adv) can induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs), which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.

Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown., (Copyright © 2013 H. Mizuguchi. Published by Elsevier B.V. All rights reserved.)

A major drawback of adenovirus (Ad) vectors is their nonspecific transduction into various types of cells or tissue after in vivo application, which might lead to unexpected toxicity and tissue damage. To overcome this problem, we developed a fiber-mutant Ad vector displaying a monobody specific for epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor 2 (VEGFR2) in the C-terminus of the knobless fiber protein derived from T4 phage fibritin. A monobody, which is a single domain antibody mimic based on the tenth human fibronectin type III domain scaffold with a structure similar to the variable domains of antibodies, would be suitable as a targeting molecule for display on the Ad capsid proteins because of its highly stable structure even under reducing conditions and low molecular weight (approximately 10 kDa). Surface plasmon resonance (SPR) analysis revealed that the monobody-displaying Ad vector specifically bound to the targeted molecules, leading to significant increases in cellular binding and transduction efficiencies in the targeted cells. Transduction with the monobody-displaying Ad vectors was significantly inhibited in the presence of the Fc-chimera protein of EGFR and VEGFR2. This monobody-displaying Ad vector would be a crucial resource for targeted gene therapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An easy and sensitive detection method for circulating tumor cells (CTC) is expected to be developed because CTC are a promising biomarker for early diagnosis of tumors and prognosis prediction of tumor patients. Our group has already developed a CTC detection method using a conditionally replicating adenovirus (Ad) which efficiently replicates and expresses GFP in telomerase-positive tumor cells. However, malignant tumor cells express much low levels of coxsackievirus and adenovirus receptor (CAR), leading to inefficient infection with a conventional conditionally replicating Ad. In addition, a tiny fraction of normal blood cells, including lymphocytes, express GFP following infection. To overcome these problems, we have developed a next-generation conditionally replicating Ad. The next-generation conditionally replicating Ad possesses the fiber protein derived from Ad serotype 35, leading to efficient infection in both CAR-positive and -negative tumor cells, because the fiber protein of Ad serotype 35 binds to CD46, which is expressed on almost all human cells. Furthermore, sequences complementary to blood cell-specific miRNA (miR-142-3p) were inserted into the 3' untranslated region of the E1 gene and GFP gene, leading to the suppression of GFP expression in normal blood cells. In this symposium, we will not only introduce the importance of CTC as a biomarker and conventional CTC detection methods but also show our data of the novel CTC detection using the next-generation conditionally replicating Ad.

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.

Pre-existing anti-adenovirus neutralizing antibodies (AdNAbs) are a major barrier in clinical gene therapy using adenovirus vectors; however, the transduction profile of adenovirus vectors in the presence of AdNAbs following intratumoral injection has not been fully examined, although such vectors are often intratumorally injected in clinical studies. In this study, we evaluated the correlation between the titer of AdNAbs in the serum and the transduction profiles in the tumor and the liver following intratumoral administration into mice possessing various titers of AdNAbs. Adenovirus vector-mediated transduction in the tumor was inhibited by AdNAbs; however, when the titer of AdNAbs was less than 200, the levels of inhibition in the transduction efficiencies within the tumor ranged from approximately 2- to 100-fold. A more than 2500-fold reduction of adenovirus vector-mediated transduction was found in most of the mice when the titers of AdNAbs were >200. On the other hand, the transduction efficiencies in the liver were largely reduced almost to the levels of the mock-transduced mice even at the low titers of AdNAbs. These results provide crucial information for the clinical use of adenovirus vectors.

Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121-1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.

Replication-incompetent adenovirus (Ad) vectors are widely used in gene therapy studies because they are beneficial as a gene delivery vehicle enabling high-titer production and highly efficient gene transfer into a wide spectrum of dividing and non-dividing cells in vitro and in vivo. Theoretically, Ad genes should not be expressed following transduction with a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with a conventional Ad vector, which leads to a cellular immunity against Ad proteins as well as Ad protein-induced toxicity. Such Ad protein-induced cellular immunity and toxicity frequently cause both an elimination of Ad vector-transduced cells and tissue damage, leading to short-lived transgene expression. To date, no detailed analysis of the leaky expression profile of Ad genes has been performed. First, we systematically examined the expression profiles of Ad genes in cells using real-time RT-PCR following transduction with a conventional Ad vector. The results revealed that significant expression was found for E2A, E4, and pIX genes. Next, in order to suppress the leaky expression of Ad genes, complementary sequences for microRNA (miRNA) were inserted into the 3'-untranslated region of the E2A, E4, or pIX genes. miRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. Incorporation of the miRNA-targeted sequences significantly suppressed the leaky expression of Ad genes in an miRNA-dependent manner.

A major limitation of the use of adenovirus (Ad) vectors is the innate immune response, which causes inflammatory cytokine production and tissue damages. To overcome this limitation, it is necessary to develop safer Ad vectors that are less likely to induce innate immunity. The Ad genome encodes two non-coding small RNAs, virus-associated (VA)-RNA I and VA-RNA II, which are transcribed by RNA polymerase III and promote Ad replication. Recently, we reported that VA-RNAs are produced in the cells transduced with a conventional first-generation (E1-deleted) Ad vector (FG-Ad) and trigger innate immune responses through intracellular nucleic acid sensors. In the present study, we have developed a VA-RNA-deleted Ad (AdΔVR) vector, in which the transcriptional control elements of the VA-RNA-expression were deleted. Although conventional HEK293 cells did not support the propagation of the AdΔVR vectors, HEK293 transformants inducibly expressing VA-RNA I (VR293 cells) with appropriate induction of VA-RNA I expression allowed the propagation of the AdΔVR vector. The AdΔVR vector showed high transduction efficiency comparable to that of the conventional FG-Ad vector in the cultured cells. The AdΔVR vector may be a safer alternative to the FG-Ad vector., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Theoretically, adenovirus (Ad) genes should not be expressed following transduction with a replication-incompetent Ad vector because the E1A gene, which is essential for the expression of other viral gene, is deleted in a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with an E1-deleted Ad vector, leading to an induction of cellular immunity against Ad proteins. To date, no detailed analysis of the leaky expression profiles of Ad genes has been performed. In this study, we systematically examined the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector (Ad-L2) at multiplicities of infection (MOIs) of 10 and 100 using real-time RT-PCR. Significant expression was found for the E4 and pIX genes following transduction with Ad-L2 in cultured cells. The expression levels of the E4 and pIX genes were approximately 30- to 600-fold lower than those of the transgene (firefly luciferase), and 50- to 5000-fold lower than those of the E4 and pIX genes following transduction at the same MOI with the wild-type Ad. Unexpectedly, expression levels of the major capsid proteins were approximately the same as, or even slightly above, the background levels (Ad gene expression levels in mock-transduced cells). This study provides valuable information for the design of a safe and efficient replication-incompetent Ad vector.

Viral vectors expressing short hairpin RNA (shRNA) are attractive for efficient and tissue-specific RNA interference (RNAi) delivery. We and others previously reported that recombinant adenovirus (Ad) vector-mediated RNAi has great potential for a variety of applications in molecular biology studies and gene therapy. In the present study, we have developed an efficient Ad vector-mediated RNAi system, in which an Ad vector carries four shRNA-expression cassettes (Ad-multi-shRNA vector), a simple and effective strategy for enhancing the RNAi response per Ad vector particle. The data demonstrated that the Ad-multi-shRNA vectors showed an enhanced RNAi effect compared to conventional Ad vectors containing a single shRNA-expression cassette. An application of the Ad-multi-shRNA vector carrying four same shRNA-sequences against the RET finger protein, an oncogene known to desensitize cells to oxidative stress and cisplatin, resulted in an enhanced cytotoxic effect of cisplatin, demonstrating the advantages of the Ad-multi-shRNA vector for silencing target genes. Furthermore, an Ad-multi-shRNA carrying four different shRNA-sequences efficiently silenced the multiple target genes simultaneously. These data suggest the potential usefulness of the Ad-multi-shRNA vector not only in basic research but also in clinical gene therapy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad E1 DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Among viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a part of a thorough evaluation of Ad-RGD in preclinical studies, we designed an experiment to investigate in detail the distribution of Ad-RGD compared with conventional adenovirus vector (WT-Ad) in pregnant mice. Surprisingly, Ad-RGD had substantial placental tropism, at 10-100 times that of WT-Ad. Transgene expression was sustained for at least 7 days, and Ad-RGD expressing firefly luciferase or red fluorescent protein has so far caused no placental dysfunction leading to fetal death. Ad-RGD showed high levels of transduction efficiency in in vitro-differentiated trophoblast stem cells, in which higher expression of αvβ3 integrin than in undifferentiated cells was observed. Our results suggest that the use of Ad-RGD or another RGD-mediated targeting strategy holds promise for drug delivery to the placenta., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Oncolytic adenoviruses (Ad) have been actively pursued as potential agents for cancer treatment. Among the various types of oncolytic Ads, the telomerase-specific replication-competent Ad (TRAD), which possesses an E1 gene expression cassette driven by the human telomerase reverse transcriptase promoter, has shown promising results in human clinical trials; however, the E1 gene is also slightly expressed in normal cells, leading to replication of TRAD and cellular toxicity in normal cells., Experimental Design: To overcome this problem, we utilized a microRNA (miRNA)-regulated gene expression system. Four copies of complementary sequences for miR-143, -145, -199a, or let-7a, which have been reported to be exclusively downregulated in tumor cells, were incorporated into the 3'-untranslated region of the E1 gene expression cassette., Results: Among the TRAD variants (herein called TRADs) constructed, TRADs containing the sequences complementary to miR-143, -145, or -199a showed efficient oncolytic activity comparable to the parental TRAD in the tumor cells. On the other hand, replication of the TRADs containing the miRNA complementary sequences was at most 1,000-fold suppressed in the normal cells, including primary normal cells. In addition, to suppress the replication of the TRADs in hepatocytes as well as other normal cells, we constructed a TRAD containing 2 distinct complementary sequences for miR-199a and liver-specific miR-122a (TRAD-122a/199aT). TRAD-122a/199aT exhibited more than 10-fold reduction in viral replication in all the normal cells examined, including primary hepatocytes., Conclusions: This study showed that oncolytic Ads containing the sequences complementary to normal cell-specific miRNAs showed significantly improved safety profiles without altering tumor cell lysis activity., (©2011 AACR.)

PEGylation of recombinant adenovirus (Ad) vectors is a promising approach for not only evasion from neutralizing anti-Ad antibodies and uptake by phagocytic cells, but also prolongation of the blood retention time of Ad vectors after systemic administration. However, the conventional PEGylation leads to significant reduction in the transduction activity of Ad vectors, probably because PEG is nonspecifically conjugated to the Ad capsid protein and inhibits the binding of Ad vectors to the primary receptor, coxsackievirus-adenovirus receptor (CAR). In order to PEGylate an Ad vector without significant reduction in the transduction activity, the biotin-binding peptide (BAP) was inserted into the hypervariable region (HVR) 5 of the hexon, which is not involved in the binding to CAR, and PEG was then specifically conjugated to the hexon HVR5 via avidin-biotin interaction. In vitro transduction experiments demonstrated that the hexon-specific PEGylation did not cause an apparent reduction in the transduction efficiency of the Ad vector, although the insertion of the BAP into the HVR5 itself reduced the transduction efficiency by 50-fold, compared with the conventional Ad vector, in the absence of anti-Ad serum. In the presence of anti-Ad serum, the transduction with the Ad vector with the BAP in the hexon HVR5 was significantly blocked; however, anti-Ad serum only slightly inhibited the transduction with the hexon-specifically PEGylated Ad vector (Ad-BAP/Bio/Avi/Bio-PEG-L2). Intravenous administration of Ad-BAP/Bio/Avi/Bio-PEG-L2 resulted in prolonged blood retention, significant reduction in the transduction in the liver, and accumulation in the tumor; however, unexpectedly, the transduction efficiency of Ad-BAP/Bio/Avi/Bio-PEG-L2 in the tumor was almost at the background level., (2010 Elsevier Ltd. All rights reserved.)

Fiber-substituted Ad serotype 5 vectors containing the fiber protein from Ad serotype 35 (Ad5F35) exhibit properties that render them suitable as a platform for targeted Ad vectors. Ad5F35 vectors do not show apparent tropism in certain organs, including the liver, and they elicit less innate immunity than other vectors after intravenous administration. In order to develop a targeted Ad vector, we previously developed fiber-mutant Ad5F35 vectors containing the integrin binding Arg-Gly-Asn (RGD) motif in the FG or HI loop of the Ad35 fiber knob. Mutant Ad5F35 vectors containing the RGD peptide in the FG or HI loop transduced CD46-negative cells more efficiently in an RGD-dependent manner, as compared to the efficiency achieved with unmodified Ad5F35 vectors (Matsui et al., 2009. Gene Therapy 16, 1050-1057). However, the transduction efficiency of the mutant Ad5F35 vectors in CD46-negative cells remained lower than had been expected. Ad5F35 vectors containing the RGD peptide in the HI or FG loop enabled a 6-fold higher transduction efficiency than that achieved with unmodified Ad5F35 vectors in CD46-negative cells, although this cell type abundantly expresses α(v)-integrins. In the present study, we aimed to enhance the transduction efficiency of fiber-mutant Ad5F35 vectors. To this end, we developed an Ad5F35-vector system in which foreign peptides could be incorporated into regions of FG and HI loops of the Ad35 fiber knob by means of in vitro ligation. Using this Ad5F35-vector system, firefly luciferase-expressing mutant Ad5F35 vectors containing the RGD peptides in both loops (Ad5F35-2xRGD-L2) were constructed. In CD46-negative cells, Ad5F35-2xRGD-L2 showed 12-fold and 3-fold greater transduction efficiency than unmodified Ad5F35 vectors and mutant Ad5F35 vectors containing only one copy of the RGD peptide in the FG or the HI loop. In addition, transduction with Ad5F35-2xRGD-L2 in CD46-negative cells was RGD peptide-dependent. These results indicate that fiber-mutant Ad5F35 vectors, by which foreign peptides can be simultaneously incorporated into both the FG and the HI loops of the Ad35 fiber knob, could be a promising gene delivery vehicle for various gene therapies, and could facilitate basic research efforts such as analyses of gene function., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recently, much attention has focused on replication-incompetent adenovirus (Ad) vectors containing fiber proteins derived from species B Ad serotype 35 (Ad35) (Ad5F35) and Ad vectors fully constructed from Ad35 as vaccine vectors expressing antigens. However, differences in the transduction properties, including the induction of innate immunity, of Ad5F35 and Ad35 vectors have not been properly and fully examined, partly because the transduction properties of these Ad vectors should be evaluated using nonhuman primates or human CD46-transgenic (CD46TG) mice, which ubiquitously express the primary receptor of Ad35, human CD46, in a pattern similar to that of humans. In the present study, we evaluated innate immune responses of mouse dendritic cells (mDCs) derived from bone marrow cells of wild-type (WT) and CD46TG mice following transduction with Ad serotype 5 (Ad5), fiber-substituted Ad5F35, or Ad35 vectors. Ad5F35 and Ad35 vectors mediated more efficient transduction in mDCs derived from CD46TG mice (CD46TG-mDCs) than did Ad5 vectors. Upregulation of costimulatory molecules and inflammatory cytokine induction by Ad5F35 and Ad35 vectors were significantly higher than those by Ad5 vectors in CD46TG-mDCs. However, the induction properties of the innate immune responses were different between Ad5F35 and Ad35 vectors. Ad35 vectors induced higher levels of costimulatory molecule expression and inflammatory cytokine production than did Ad5F35 vectors in CD46TG-mDCs. Furthermore, intravenous administration of Ad35 vectors in WT and CD46TG mice resulted in higher levels of serum interleukin (IL)-6 and IL-12 compared with administration of Ad5F35 vectors, which exhibited almost mock-transduced levels of these inflammatory cytokines. This study indicates that innate immune responses by Ad35 and Ad5F35 vectors are distinct even although both Ad vectors recognize human CD46 as a receptor., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Target tissue-specific delivery and transcription of foreign genes are desirable for safe and effective gene therapy. Two approaches for this purpose, "Targeted Delivery" and "Targeted Expression", have been mainly reported. Among "Targeted Expression" approaches, microRNA (miRNA)-mediated "post-transcriptional de-targeting" has been recently demonstrated, and much attention has been focused on this approach. MiRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region (UTR) of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. First, in order to reduce the hepatic transduction by Ad vectors, complementary sequences of liver-specific miRNA, miR-122a, were inserted into the 3'-UTR of the transgene expression cassette. Intratumor injection of this Ad vector resulted in approximately 100-fold lower hepatic expression than that of the conventional Ad vector, without reducing gene expression in the tumor. Second, complementary sequences for miRNAs selectively down-regulated in tumor cells were inserted into the E1 gene expression cassette in oncolytic Ads, which exhibit tumor cell-specific replication and antitumor effects. Recent studies demonstrated that expression of several miRNAs is exclusively reduced in tumor cells. Oncolytic Ads containing the miRNA complementary sequences showed reduced replication in the normal cells, without altering the antitumor effects. MiRNA-regulated gene expression system mediates "post-transcriptional de-targeting", in which translation of transgene is suppressed in a tissue-specific manner; however, tissue-specific transgene expression can be achieved by taking tropism of gene delivery vehicles into consideration and reducing the transgene expression in untargeted organs via miRNA-regulated gene expression system.

Transduction with replication-incompetent recombinant adenovirus (Ad) vectors results in a rapid activation of innate immune responses, such as inflammatory cytokine production and subsequent tissue damage. The precise mechanisms of the innate immune responses induced by Ad vectors remain to be clarified. Possible components of Ad vectors that activate innate immune responses are the capsid protein, the viral genome (DNA), and viral transcripts. In the present study, we demonstrate that virus-associated RNAs (VA-RNAs), which are small RNAs transcribed by RNA polymerase III, induce the production of type I IFN (IFN-α and IFN-β), but they do not induce the production of inflammatory cytokines (IL-6 and IL-12), in mouse embryonic fibroblasts (MEFs) and granulocyte-macrophage colony-stimulating factor-generated bone marrow-derived dendritic cells (GM-DCs). We also show that IFN-β promoter stimulator-1 is involved in VA-RNA-dependent IFN-β production in MEFs and is partially involved in type I IFN production in GM-DCs. This study provides important insight into the mechanisms of Ad vector-triggered innate immune responses, which may lead to more advanced and rational Ad vector designs for gene therapies and vaccine applications.

We examined the transduction efficiency in human embryonic stem (ES) and induced pluripotent stem (iPS) cells using an adenovirus (Ad) vector. RT-PCR analysis revealed the expression of the coxsackievirus and adenovirus receptor, a receptor for Ad, in these cells. However, gene expression after the transduction with an Ad vector was observed only in the periphery of ES and iPS cell colonies, when human ES and iPS cells were passaged as small colonies. This suggests that the Ad vector could not enter inside the ES and iPS cell colonies by their tight connection. We thus attempted to transduce foreign genes into the dissociated form of human ES and iPS cells, which were passaged using Rho-associated kinase inhibitor. In this condition, transduction efficiency in human ES and iPS cells was markedly increased and transgene expression was observed even inside the colonies by using Ad vectors. Furthermore, Ad vector-mediated transduction did not alter the expression of undifferentiated markers such as Oct-3/4, Nanog, and SSEA-4. Our results indicate that Ad vectors are effective tools for transduction into human ES and iPS cells.

Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury; however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase, which is an antioxidant enzyme scavenging H(2)O(2), resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model, CCl(4)-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing beta-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl(4)-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury., ((c) 2009. Published by Elsevier B.V. All rights reserved.)

Fiber-substituted adenovirus (Ad) vectors containing fibers of Ad serotype 35 (AdF35) efficiently transduce a variety of human cells because their receptor, human CD46, is ubiquitously expressed on almost all nucleated cells. However, the ubiquitous expression of CD46 might lead to unexpected transduction in untargeted organs. In this study, we developed fiber-modified AdF35 vectors with an integrin-binding Arg-Gly-Asn (RGD) peptide incorporated into the FG, HI or IJ loop, which have been identified as important regions for binding to CD46. Incorporation of foreign peptides into these loops does not inhibit trimerization of the fibers. In CD46-negative cells, fiber-mutant AdF35 vectors containing an RGD peptide in the FG or HI loop showed 6- to 30-fold higher transduction efficiencies in an RGD-peptide-dependent manner than the unmodified AdF35 vectors. In contrast, in CD46-positive cells, insertion of foreign peptides markedly reduced the transduction efficiencies of the AdF35 vectors, indicating that insertion of foreign peptides significantly inhibits binding to CD46. In particular, CD46-mediated transduction was completely diminished by insertion of foreign peptides into the HI loop. Our findings indicate that HI loop is the most suitable domain to mediate a foreign peptide-dependent and CD46-independent transduction by incorporation of foreign peptides into the Ad35 fiber knob.

Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.

To date, no countermeasures have been developed to overcome adenovirus (Ad) vector-induced innate immune responses in gene therapies that utilize these vectors. In the present study, we attempted to suppress Ad vector-induced innate immune responses and hepatotoxicity by delivering an NF-kappaB decoy to splenic macrophages and dendritic cells, as well as to Kupffer cells in the liver, using sugar-modified cationic liposomes. Pre-injection of the sugar-modified cationic liposome/NF-kappaB decoy complexes reduced serum levels of interleukin (IL)-12, which is a typical inflammatory cytokine induced by Ad vectors, and also reduced hepatotoxicity following Ad vector injection. Electrophoretic mobility shift assay demonstrated a reduction of Ad vector-induced NF-kappaB activation by pre-injection of the sugar-modified cationic liposome/NF-kappaB decoy complexes. Two types of sugar-modified cationic liposomes, fucosylated cationic liposomes (Fuc-liposomes) and mannosylated cationic liposomes (Man-liposomes), were tested in this study. Man-liposomes, which are considered to have greater affinity than Fuc-liposomes for splenocytes, appeared to have superior suppressive effects to those of Fuc-liposomes. Pre-injection of the sugar-modified cationic liposome/NF-kappaB decoy complexes did not inhibit Ad vector-mediated transduction in the organs. This study provides important findings for overcoming Ad vector-induced innate immune responses.

Properties of gene delivery vehicles, including gene transfer efficiencies and toxicities, are a key parameter for successful gene therapy. Among various types of gene delivery vehicles that have been developed so far, adenovirus (Ad) vectors have promising potentials as a vector for gene therapy because they can easily be grown to high titers and can efficiently deliver genes to both dividing and non-dividing cells. However, recent studies demonstrated some drawbacks of conventional Ad vectors, which are composed of subgroup C Ad serotype 5 (Ad5). First, Ad5 vectors poorly transduce cells lacking the primary receptor for Ad5, coxsackievirus and adenovirus receptor (CAR). Second, majority of adults have neutralizing antibodies to Ad5. In order to overcome these drawbacks, we developed a novel Ad vector which is fully composed of subgroup B Ad serotype 35 (Ad35). Ad35 vectors can infect a variety of human cells because the primary receptor for Ad35, CD46, is ubiquitously expressed in human cells. Furthermore, Ad35 vectors efficiently transduce in the presence of anti-Ad5 antibodies, and seroprevalence of Ad35 in adults is much lower than that of Ad5. In the current review, I introduce our recent work on development and evaluation of Ad35 vectors, and I also discuss the potential of Ad35 vectors as gene delivery vehicles.

The capacity of gene delivery vehicles is considered to be a critical factor determining the success of gene therapy. To date, various types of gene delivery vehicle have been developed. Among them, recombinant adeno-virus (Ad) vectors have potential that has favored their worldwide use in vitro and in vivo. Conventional Ad vectors are composed of subgroup C Ad serotype 5 (Ad5), although it has been clarified that the drawbacks of Ad5 vectors are a high seroprevalence of Ad5 in adults and low transduction efficiencies in cells lacking the primary receptor for Ad5, coxsackievirus and adenovirus receptor. To overcome these problems, we developed a novel Ad vector fully composed of Ad serotype 35 (Ad35). Ad35 vectors show a wide tropism for human cells because Ad35 binds to human CD46, which is ubiquitously expressed on almost all human cells, as a primary receptor. In addition, anti-Ad5 antibodies do not inhibit Ad35 vector-mediated transduction and the seroprevalence of Ad35 in adults is lower than that of Ad5. This paper reviews our studies on the development and evaluation of Ad35 vectors. Ad vectors derived from other Ad serotypes different from Ad5, including Ad35, are expected to be gene delivery vehicles alternative to conventional Ad5 vectors.

Mast cells (MCs) have been shown to play an important role in immunoglobulin E (IgE)-associated immediate hypersensitivity and innate immunity by producing a variety of lipid mediators and cytokines. An efficient gene-delivery system is indispensable for elucidation of these mechanisms. In the present study, human and rodent MCs were transduced with various types of modified adenovirus (Ad) vectors. Fiber modification in Ad vectors significantly improved the transduction efficiencies in MCs. A fiber-substituted Ad serotype 5 (Ad5) vector containing Ad serotype 35 (Ad35) fiber proteins (Ad5F35) and an Ad35 vector, both of which transduce cells via human CD46, mediated 9.9-fold and 10.1-fold higher transduction efficiencies than conventional Ad5 vectors in the human mast cell line LAD2 among the Ad vectors. Ad5F35 and Ad35 vectors also efficiently transduced bone marrow-derived MCs (BMMCs) prepared from human CD46-transgenic (CD46TG) mice. The rat mast cell line RBL-2H3 were most efficiently transduced with a fiber-mutant Ad5 vector containing the Arg-Gly-Asp (RGD) peptide in the HI loop (Ad-RGD) of the fiber knob. Transduction with the Ad vectors did not induce degranulation or inflammatory cytokine production in the MCs. These results indicate that Ad vectors, including fiber-mutant Ad vectors, are effective gene-delivery tools for MCs.

Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35DeltaRGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The alphav-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-alpha, and interferon-alpha in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of alphav-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.

Adenovirus (Ad) vector application in gene therapy is limited by its naïve tropism. We previously developed protein IX (pIX)-modified and hexon-modified Ad vectors in order to alter Ad vector tropism. However, these modified Ad vectors failed to infect cells with the foreign ligands displayed in the pIX or hexon. We hypothesized that steric hindrance by fiber proteins might have prevented the ligand-mediated transduction, as fibers are the outmost capsid proteins of Ad vectors. Therefore, we generated a series of fiberless Ad vectors and investigated their gene expression properties. Unexpectively, however, pIX- or hexon-modified fiberless Ad vector did not achieve any gene expression (the gene expression level by these vectors was similar to the background level). These results might be caused by the fact that the fiberless particles were weaker against physical burdens. To the best of our knowledge, this study is the first reported attempt to develop fiberless Ad vectors containing foreign ligands in the pIX or hexon region. The drawback of the lower stability of fiberless Ad vectors must be overcome to develop targeted Ad vectors based on such vectors. This study could provide basic information for the development of effective targeted Ad vectors.

Adenovirus serotype 35 (Ad35) vectors have shown promise as effective gene delivery vehicles. However, the transduction profiles of Ad35 vectors in conventional mice allow only a limited estimation of transduction properties of these vectors, because the mouse analog of the subgroup B Ad receptor, CD46, is restricted to the testis. In order to assess the transduction properties of Ad35 vectors more completely, we performed transduction experiments using cynomolgus monkeys, which ubiquitously express CD46 in a pattern similar to that in humans. In vitro transduction experiments demonstrated that cultured cells from the cynomolgus monkey were efficiently transduced with Ad35 vectors. In contrast, after intravenous administration into live monkeys hardly any evidence of Ad35 vector-mediated transduction was found in any of the organs, although Ad35 vector genomes were detected in various organs. Less severe histopathological abnormalities were found in the Ad35 vector-infused monkeys than in the conventional Ad5 vector-injected monkeys. In the latter, serious tissue damage and inflammatory responses, such as hepatocyte necrosis and lymphatic hyperplasia in the colon, were induced. Both Ad35 and Ad5 vectors caused similar hematological changes (increase in CD3(+) cells, and decrease in CD16(+) cells and CD20(+) cells) in peripheral blood cells. These results should provide valuable information for the clinical application of Ad35 vectors.

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.

Most subgroup B adenoviruses (Ads), including adenovirus (Ad) serotype 35 (Ad35), bind to human CD46 as a receptor; however, the infection processes of subgroup B Ads following attachment to CD46 remain to be elucidated. Subgroup B Ads possess Arg-Gly-Asp (RGD) motifs in the penton base, similarly to subgroup C Ad serotypes 2 and 5. In this study, we examined the role of penton base RGD motifs in Ad35 vector-mediated transduction in human hematopoietic cells. Inhibition of interaction between integrins and the RGD motifs by divalent cation chelation and a synthetic RGD peptide reduced the transduction efficiencies of Ad35 vectors; however, the amounts of cell-associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not decreased by divalent cation chelation or the RGD peptide. Mutation of penton base RGD motifs reduced the transduction efficiencies of Ad35 vectors, although the amounts of cell-associated vector DNA of Ad35 vectors at 4 or 37 degrees C were not altered by mutation of penton base RGD motifs in Ad35 vectors. Furthermore, preincubation with several types of anti-integrin antibodies significantly inhibited Ad35 vector-mediated transduction. These results suggest that interaction between integrins and penton base RGD motifs plays a crucial role in Ad35 vector-mediated transduction in hematopoietic cells, probably in the post-internalization steps.

Conjugation of polyethylene glycol to protein or particles (PEGylation) prolongs their plasma half-lives and promotes their accumulation in tumors due to enhanced permeability and retention (EPR) effect. Although PEGylation of adenovirus vectors (Ads) is an attractive strategy to improve the in vivo kinetics of conventional Ads, the EPR effect of PEGylated Ad (PEG-Ad) had not previously been reported. In this study, we prepared PEG-Ads with PEG at various modification ratios, injected them intravenously into tumor-bearing mice, and determined the blood kinetics, viral distribution, and gene expression patterns, respectively. In addition, we conducted a cancer therapeutic study of PEG-Ad encoding tumor necrosis factor (TNF)-alpha. The plasma half-life of PEG-Ad was longer than that of unmodified-Ad, and accumulation of PEG-Ad in tumor tissue increased as the PEG modification ratio increased. In particular, PEG-Ad with about 90% modification ratio showed higher (35 times) gene expression in tumor and lower (6%) in liver, compared with values for unmodified Ad. Moreover, PEG-Ad encoding TNF-alpha demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with unmodified-Ad. PEGylation of Ad achieved tumor targeting through the EPR effect, and these attributes suggest that systemic injection of PEG-Ad has great potential as an anti-tumor treatment.