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Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads. [ABSTRACT FROM AUTHOR]

Oncolytic adenoviruses (OAds), most of which are based on species C human adenovirus serotype 5 (Ad5) (OAd5), have recently received much attention as potential anticancer agents. High seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based gene therapy. However, the impacts of anti-Ad5 neutralizing antibodies on OAd5-mediated transgene expression in the tumor and antitumor effects remain to be fully elucidated. In this study, we examined the impact of anti-Ad5 neutralizing antibodies on the OAd5-mediated antitumor effects and OAd5-mediated transgene expression. The luciferase expression of OAd-tAIB-Luc, which contains the cytomegalovirus promoter-driven luciferase gene, was inhibited in human cultured cells in the presence of human serum. Although the inhibitory effects of human serum possessing the low anti-Ad5 neutralizing antibody titers were overcome by long-term infection, the in vitro tumor cell lysis activities of OAd-tAIB-Luc were entirely attenuated by human serum containing the high titers of anti-Ad5 neutralizing antibodies. OAd-tAIB-Luc-mediated luciferase expression in the subcutaneous tumors 3 days after administration and tumor growth suppression levels following intratumoral administration were significantly lower in mice possessing the high titers of anti-Ad5 neutralizing antibodies, compared to those in control mice. These results suggested that pre-existing anti-Ad5 antibodies attenuated both transgene expression and potential antitumor effects of OAd5 following intratumoral administration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: We previously developed a hexon-specific PEGylated adenovirus (Ad) vector by utilizing avidin-biotin interaction. However, the Ad vector was aggregated due to the multiple interactions between avidin and biotin, resulting in a reduction in the transduction efficiencies in the organs following systemic administration. In this study, we developed a new method for hexon-specific PEGylation by mixing Ad vectors with PEGylated blood coagulation factor X (FX) (PEG-FX). FX specifically binds to the hexon protein, suggesting that FX serves as an adaptor molecule for hexon-specific modification. Intravenous administration of the PEG-FX-associated Ad (PEG-FX-Ad) vector into conventional mice resulted in prolonged blood retention. However, the transduction efficiencies in the liver were not reduced by PEG-FX. On the other hand, in the warfarinized mice, the PEG-FX-Ad vectors exhibited a significant reduction in the liver transduction. In addition, incubation of the PEG-FX-Ad vector with unmodified FX resulted in dissociation of PEG-FX from the Ad vector, indicating that a substitution of PEG-FX with endogenous FX occurs in the blood following administration. This study demonstrates that FX can be used as an adaptor molecule for hexon-specific modification; however, modified FX might be substituted with endogenous FX in the blood. [Copyright &y& Elsevier]

Abstract: Recent studies have demonstrated that the liver-specific microRNA (miRNA) miR-122a plays an important role in the replication of hepatitis C virus (HCV). Antisense nucleotides against miR-122a, including locked nucleic acid (LNA), have shown promising results for suppression of HCV replication; however, a liver-specific delivery system of antisense nucleotides has not been fully developed. In this study, an adenovirus (Ad) vector that expresses tough decoy (TuD)-RNA against miR-122a (TuD-122a) was developed to suppress the HCV replication in the liver hepatocytes. Ad vectors have been well established to exhibit a marked hepatotropism following systemic administration. An in vitro reporter gene expression assay demonstrated that Ad vector-mediated expression of TuD-122a efficiently blocked the miR-122a in Huh-7 cells. Furthermore, transduction with the Ad vector expressing TuD-122a in HCV replicon-expressing cells resulted in significant reduction in the HCV replicon levels. These results indicate that Ad vector-mediated expression of TuD-122a would be a promising tool for treatment of HCV infection. [Copyright &y& Elsevier]

The major limitation of the clinical use of replication-incompetent adenovirus (Ad) vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN), following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs). In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88) and toll-like receptor 9 (TLR9) play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs), which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1)-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs. [ABSTRACT FROM AUTHOR]

Abstract: 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 αv-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 &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 H2O2, 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, CCl4-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 β-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl4-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. [Copyright &y& Elsevier]

Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. Previously, by using an adenovirus (Ad) vector containing the elongation factor-1α (EF-1α) and the cytomegalovirus enhancer/β-actin (CA) promoters, we developed an efficient transduction system for mouse embryonic stem (ES) cells and their aggregate form, embryoid bodies (EBs). In this study, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector containing EF-1α and the CA promoter could efficiently transduce transgenes into mouse iPS cells. At 3,000 vector particles/cell, 80%-90% of iPS cells expressed transgenes by treatment with an Ad vector containing the CA promoter, without a decrease in pluripotency or viability. We also found that the CA promoter had potent transduction ability in iPS cell-derived EBs. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to increase cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells. [ABSTRACT FROM AUTHOR]

Abstract: 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-κB 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-κB 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-κB activation by pre-injection of the sugar-modified cationic liposome/NF-κB 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-κB 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. [Copyright &y& Elsevier]

The combined use of adenovirus (Ad) vectors expressing herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) offers a potential therapeutic strategy against cancer. However, intratumorally injected Ad vectors are disseminated into the systemic circulation and efficiently transduce the liver, resulting in severe hepatotoxicity. In order to overcome this problem, an Ad vector carrying a microRNA (miRNA)-regulated expression system was developed by inserting into the 3′-untranslated region (3′-UTR) of the expression cassette four tandem copies of sequences with perfect complementarity to miR-122a, which exhibits liver-specific expression. Transgene expression from the Ad vector carrying the miR-122a target sequences was 7- to 70-fold lower in cells with high miR-122a expression as compared to expression from a conventional Ad vector. Intratumoral injection of the Ad vector containing the miR-122a target sequences resulted in a 130- to 1,500-fold reduction in hepatic transgene products (without affecting the transgene expression in the tumor) when compared with those from a conventional Ad vector. In suicide gene therapy, the inclusion of the miR-122a target sequences in the HSVtk expression cassette achieved not only significant antitumor effects, but also a dramatic reduction in HSVtk/GCV-induced hepatotoxicity. These results indicate that Ad vectors that mediate miR-122a-regulated HSVtk expression provide a safe and efficient suicide gene therapy strategy.Molecular Therapy (2008) 16 10, 1719–1726 doi:10.1038/mt.2008.159 [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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.Molecular Therapy (2008); 16 4 726–733 doi:10.1038/mt.2008.19 [ABSTRACT FROM AUTHOR]

Abstract: Human CD46 (membrane cofactor protein) has recently been identified to be an attachment receptor for subgroup B adenoviruses (Ads); however, the precise interaction between human CD46 and subgroup B Ads are just beginning to be understood. In this study, to characterize the interaction between human CD46 and subgroup B Ads, varieties of mutant CD46 were tested for their ability to act as a receptor for Ad serotype 35 (Ad35), which belongs to subgroup B. In addition, we determined Ad35 vector-mediated transgene expression and cellular uptake of Ad35 vectors in the presence of a set of anti-CD46 antibodies. Our data demonstrated that the short consensus repeats (SCRs) 1 and 2 in human CD46 are important for interaction with Ad35, whereas the cytoplasmic domain of human CD46 was found not to be required for the function as an Ad35 receptor. Rather, a complete deletion of the cytoplasmic domain of human CD46 increased the transduction efficiencies of Ad35 vectors. This information should help in elucidation of the mechanism of subgroup B Ad infection, as well in the improvement of the subgroup B Ad vectors. [Copyright &y& Elsevier]

We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector. [Copyright &y& Elsevier]

Because of their capacity for self-renewal and multilineage differentiation into all types of mature blood cells, hematopoietic stem cells (HSCs) are highly attractive, not only as targets of gene therapy but also as cell sources for regeneration medicine. Hence, efficient transduction into HSCs holds promise for treating a number of diseases that result from abnormal blood cell function, and it would provide a powerful approach for understanding their basic biology and for manipulating their properties in vitro and in vivo. Numerous studies of transduction into HSCs have been performed using various types of gene transfer vectors. Retrovirus vectors and lentivirus vectors are often used for transduction into HSCs when stable transgene expression is desirable. In contrast, adenovirus (Ad) vectors are highly suitable when persistent gene expression followed by integrating viral vector-mediated gene transfer is not appropriate, for example, in ex vivo manipulation of HSCs and functional analysis of genes of interest in HSCs. We previously developed a novel type of Ad vector, composed of whole Ad serotype 35 (Ad35) belonging to subgroup B, and demonstrated that Ad35 vectors gave efficient gene transfer into human bone marrow CD34+ cells (Gene Ther. 2003, 10, 1041–1048; Gene Ther. 2005, 12, 1424–1433). However, rates of Ad35 vector-mediated transduction into mouse HSCs are relatively low because a receptor for Ad35, CD46, is not expressed in mouse HSCs. Because the cell fraction containing mouse HSCs is well characterized and the biology of the mouse HSC has been studied extensively in mouse models, efficient transduction with Ad vectors into mouse HSCs would be useful. Here, we performed Ad35 vector-mediated transduction into mouse HSCs isolated from human CD46-transgenic (CD46TG) mice, which express human CD46 in a pattern similar to that in humans. First, bone-marrow- derived C-kit(+)Lineage(−) (KL) cells were recovered from wild- type mice and homozygous CD46TG mice as mouse HSCs, and human CD46 expression in KL cells was examined. More than 80% of KL cells from CD46TG mice expressed human CD46. Ad35 vector-mediated transduction demonstrated that KL cells from wild- type mice were refractory to Ad35 vectors; in contrast, KL cells from CD46TG mice showed high levels of transgene expression. The human elongation factor 1α promoter (EF1α promoter) worked most efficiently of all the promoters examined in KL cells. In addition, colony-forming assay revealed that Ad35 vector-mediated transgene-expressing KL cells from CD46TG mice differentiated into progenitor cells of all lineages. These results suggest that this model using Ad35 vector and KL cells from CD46TG mice would be a powerful tool for the study of gene function in mouse HSCs.Molecular Therapy (2006) 13, S52–S53; doi: 10.1016/j.ymthe.2006.08.150 [ABSTRACT FROM AUTHOR]

An abstract of the article "Optimization of Adenovirus Serotype 35 Vectors for Efficient Transduction in Human Hematopoietic Progenitors; Comparison of Promoter Activities," by Fuminori Sakurai, Hiroyuki Mizuguchi and Kenji Kawa is presented.

[Display omitted] Adenovirus (Ad) vectors based on human adenovirus serotype 5 (Ad5) have attracted significant attention as vaccine vectors for infectious diseases. However, the effectiveness of Ad5 vectors as vaccines is often inhibited by the anti-Ad5 neutralizing antibodies retained by many adults. To overcome this drawback, we focused on human adenovirus serotype 35 (Ad35) vectors with low seroprevalence in adults. Although Ad35 vectors can circumvent anti-Ad5 neutralizing antibodies, vector yields of Ad35 vectors are often inferior to those of Ad5 vectors. In this study, we developed novel Ad35 vectors containing the Ad5 E4 orf 4, 6, and 6/7 or the Ad5 E4 orf 6 and 6/7 for efficient vector production, and compared their properties. These E4-modified Ad35 vectors efficiently propagated to a similar extent at virus titers comparable to those of Ad5 vectors. An Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 mediated more efficient transduction than that containing the Ad5 E4 orf 6 and 6/7 in human cultured cells. Furthermore, insertion of an arginine-glycine-aspartate (RGD) peptide in the fiber region of an Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 significantly improved the transgene product-specific antibody production following intramuscular administration in mice. The Ad35 vector containing the RGD peptide mediated efficient vaccine effects even in the mice pre-immunized with an Ad5. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: We investigated the efficiency of gene transduction into murine pancreatic islets using the adenovirus (Ad) vector. Western blotting analysis showed that mouse pancreatic islets express coxsackievirus and adenovirus receptor, a receptor for Ad. Nevertheless, gene expression after transduction of the Ad vector in vitro was observed only in the periphery of the islets, probably due to physical obstruction against Ad infection of the cells in the inside of islets. Ca2+-free treatment before the Ad vector transduction enhanced transduction efficiency in the islets, but not the cells in the inside of islets. The Ad vector transduction through the celiac artery in vivo and then cultivation of islets in vitro resulted in efficient transduction even in the inside of islets. Thus we propose a new strategy for efficient gene transfer to pancreatic β-cells. [Copyright &y& Elsevier]

Abstract: Previously, we developed single adenovirus (Ad) vectors that contained the gene of interest in the E1 deletion region and the transactivator gene for the tetracycline-controllable expression system in the E3 deletion region. In the present study, we improved the Ad vector-mediated tetracycline-controllable expression system by the fiber modification of Ad. We developed fiber-modified Ad vectors containing the tet-off system, which are effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Ad vectors containing the tet-off system with an Arg–Gly–Asp (RGD) peptide in the HI loop of the fiber knob or the Ad type 35 fiber greatly improved transduction efficiency (more than 1–2-log orders) into the cells lacking CAR expression but expressing αv integrin or CD46, respectively. They exhibited vastly higher regulation of gene expression by doxycycline. The combination of fiber-modified Ad vectors and the tetracycline-controllable expression system should offer a powerful tool for gene therapy and gene transfer experiment. [Copyright &y& Elsevier]

Abstract: Adenovirus (Ad) vectors have been expected to play a great role in gene therapy because of their extremely high transduction efficiency and wide tropism. However, due to the intrinsic deficiency of their immunogenic toxicities, Ad vectors are rapidly cleared from the host, transgene expression is transient, and readministration of the same serotype Ad vectors is problematic. As a result, Ad vectors are continually undergoing refinement to realize their potential for gene therapy application. Even after 1999, when a patient fatally succumbed to the toxicity associated with Ad vector administration at a University of Pennsylvania (U.S.) experimental clinic, enthusiasm of gene therapists for Ad vectors has not waned. With great efforts from various research groups, significant advances have been achieved through comprehensive approaches to improving the kinetics of Ad vector-delivered genes and gene products. [Copyright &y& Elsevier]

The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triplemutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors. [ABSTRACT FROM AUTHOR]

Abstract: Adenovirus vector (Adv) vaccination at a systemic site, such as intramuscular (i.m.) immunization, can induce antigen-specific CD8+ T cell responses in both systemic and mucosal compartments. It remains unclear, however, how antigen-specific CD8+ T cell response is induced in the mucosa. In this study, we found that type-I IFN signaling is required for the induction of mRNA expression of retinal dehydrogenase in the draining lymph nodes following the i.m. Adv vaccination. We show that type-I IFN signaling is required for the induction of antigen-specific CD8+ T cell response in the gut-mucosal compartment following the i.m. Adv vaccination. [Copyright &y& Elsevier]

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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% (37.7kb) of the wild-type genome (35.9kb) 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 37.7kb 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% (38.9kb) 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 &y& Elsevier]

Abstract: Bone marrow stromal cells (BMSCs) are expected to be a source for tissue regeneration because they can differentiate into multiple cell types. Establishment of efficient gene transfer systems for BMSCs is essential for their application to regenerative medicine. In this study, we compared the transduction efficiency in mouse primary BMSCs by using fiber-modified adenovirus (Ad) vectors, and demonstrated that AdK7, which harbors a polylysin (K7) peptide in the C-terminus of the fiber knob, could efficiently express a transgene in BMSCs. Notably, AdK7 robustly drove transgene expression in more than 90% of the BMSCs at 3,000 vector particles/cell. Furthermore, we showed that in vitro and in vivo osteogenic potential of BMSCs was dramatically promoted by the transduction of Runx2 gene using AdK7. These results indicate that this transduction system could be a powerful tool for therapeutic applications based on BMSCs. [Copyright &y& Elsevier]

Abstract: Human mesenchymal stem cells (hMSCs) are considered a source of cells for regenerative medicine, and cell and gene therapy. Efficient gene transfer into hMSCs is essential for basic investigations into cellular differentiation and developmental biology, and for therapeutic applications in gene-modified regenerative medicine. In the present study, we optimized the transduction of hMSCs by means of fiber-modified adenovirus (Ad) vectors. Among the various types of Ad vectors tested, the polylysine modification of the C-terminal of the fiber knob most markedly improved the efficiency of hMSC transduction. At 300 vector particles per cell of polylysine-modified Ad vectors, more than 95% of the hMSCs expressed transgene. In this condition, polylysine-modified Ad vectors mediated 460-fold more transgene activity than the conventional Ad vectors. Ad vectors containing the Ad type 35 fiber or an Arg-Gly-Asp (RGD) peptide in the fiber knob mediated 130 or 16 times, respectively, the transgene activity mediated by the conventional Ad vectors. We also examined the efficiency of transduction into adipogenic-differentiated hMSCs. In this latter case, only Ad vectors containing the Ad type 35 fiber showed efficient gene expression. These results showed that fiber-modified Ad vectors could become a potent tool for basic research into, and the therapeutic application of, hMSCs and adipogenic-differentiated hMSCs. [Copyright &y& Elsevier]

Adenovirus (Ad) vectors derived from Ad type 5 are widely used in gene therapy and infect a broad range of cells. One of the limitations of Ad vector-mediated gene transfer is the innate immune response after systemic injection of the vector. Ad vectors induce the expression of numerous inflammatory cytokines and chemokines, such as interleukin 6 (IL-6), IL-12, tumor necrosis factor a (TNF-α), interferon-γ-inducible protein 10 (IP-10), RANTRS (regulation on activation, normal T cell expressed and secreted), monocyte chemoattractant protein 1 (MCP-1) et al. Ad particle or capsid proteins, not viral transcription products, trigger this immune response. The strategy to blunt (or reduce) this innate immune response or the development of Ad vectors with reduced innate immune response as well as the elucidation of mechanism of Ad vector-mediated innate immune response are required for safe gene therapy using Ad vectors. In the present study, we developed the modified Ad vectors with reduced innate immune response.First, we examined the organs responsible to secret cytokines after the systemic injection of Ad vectors into mouse. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine (IL-6, IL-12, TNF-α) expression. Then, we examined the transgene expression in each organs, tissue distribution of Ad vector DNA, the serum cytokine levels, and serum ALT levels (as a marker of liver toxicity) after the injection of several types of capsid-modified Ad vectors in comparison with the conventional Ad vector. One of the modified Ad vectors showed higher transgene expression in each organs including liver than the conventional Ad vector. Interestingly, this Ad vector showed much reduced serum IL-6 and ALT levels, which were almost background levels. Quantitative PCR analysis to examine tissue distribution of Ad vector DNA showed that spleen distribution was reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. There was no difference of serum IL-12 levels between this modified Ad vector and the conventional Ad vector. Therefore, IL-6 and IL-12 would be expressed in different mechanism or different types of cells. We now examine the cell types responsible to secret cytokine expression in the spleen and other cytokines and chemokines expression in each organ by RT-PCR analysis.Molecular Therapy (2006) 13, S57–S58; doi: 10.1016/j.ymthe.2006.08.167 [ABSTRACT FROM AUTHOR]

An abstract of the article "Reductions in Tissue Transduction and Toxicity after Systemic Administration of Adenovirus Vectors Containing Fiber-Shaft Exchange in Combination with both CAR- and av Integrin-Binding Ablation," by Naoya Koizumi and colleagues is presented.