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Previous studies have shown promising results when using an agonistic anti-4-1BB antibody treatment against established tumors. While this is promising, this type of treatment can induce severe side effects. Therefore, we decided to incorporate the membrane form of 4-1BB ligand (4-1BBL) in a replicative deficient adenovirus vaccine expressing the invariant chain (Ii) adjuvant fused to a tumor associated antigen (TAA). The Ii adjuvant increases and prolongs TAA specific CD8+ T cells as previously shown and local expression of 4-1BBL was chosen to avoid the toxicity associated with systemic antibody administration. Furthermore, adenovirus encoded 4-1BBL expression has previously been successfully used to enhance responses toward Plasmodium falciparum and Influenza A antigens. We showed that the incorporation of 4-1BBL in the adenovirus vector led to surface expression of 4-1BBL on antigen presenting cells, but it did not enhance T cell responses in mice towards the Ii linked antigen. In tumor-bearing mice, our vaccine was found to decrease the frequency of TAA specific CD8+ T cells, but this difference did not alter the therapeutic efficacy. In order to reconcile our findings with the previous reports of increased anti-cancer efficacy using systemically delivered 4-1BB agonists, we incorporated a secreted version of 4-1BBL (Fc-4-1BBL) in our vaccine and co-expressed it with the Ii linked to TAA. In tumor bearing mice, this vaccine initially delayed tumor growth and slightly increased survival compared to the vaccine expressing the membrane form of 4-1BBL. Accordingly, secreted 4-1BBL co-encoded with the Ii linked antigen may offer a simplification compared to administration of drug and vaccine separately., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

The potential of RNA interference (RNAi)-based gene therapy has been demonstrated in many studies. However, clinical application of this technology has been hampered by a paucity of efficient and safe methods of delivering the RNAi activators. Prolonged transgene expression and improved safety of helper-dependent adenoviral vectors (HD AdVs) makes them well suited to delivery of engineered artificial intermediates of the RNAi pathway. Also, AdVs' natural hepatotropism makes them potentially useful for liver-targeted gene delivery. HD AdVs may be used for efficient delivery of cassettes encoding short hairpin RNAs and artificial primary microRNAs to the mouse liver. Methods for the characterization of HD AdV-mediated delivery of hepatitis B virus-targeting RNAi activators are described here.

Gastric cancer is a highly lethal malignancy and its 5-year survival rate remains depressed in spite of multiple treatment options. Targeting drug delivery to tumor vasculature may be a promising strategy for gastric cancer therapy, for it can block the nutrition source of tumor and inhibit the metastasis and invasion in a certain extent. In present study, we have prepared the drug-targeting delivery system of peptide GX1-mediated anionic liposomes carrying adenoviral vectors (GX1-Ad5-AL), in which the tumor suppressor gene of PTEN was integrated into DNA of Ad5 and the GX1 peptide could play targeting role to vascular of gastric cancer. The inhibition ability of GX1-Ad5-AL to human gastric cancer cell lines (SGC-7901) and human umbilical vein endothelial cells (HUVEC) was evaluated by MTT assay. Further, the cell migration assay was carried out in transwell inserts and the cells uptaking of GX1-Ad5-AL was detected by confocal laser scanning microscopy. The experimental results indicated that the average cell proliferation inhibition rates resulted from the drug delivery system of GX1-Ad5-AL in SGC-7901 and HUVEC were 68.36% and 64.13%, respectively which were higher than that resulted from GX1 or Ad5-AL. Meanwhile, results of cell migration experiment demonstrated that GX1-Ad5-AL could significantly suppress the migration of gastric cancer cell of SGC-7901. Moreover, both the imaging from confocal laser scanning microscopy and the quantitative analysis of fluorescence intensity showed that, GX1-Ad5-AL was more easily uptaken by SGC-7901 cells, as compared to Ad5-AL. Therefore, the formulation of GX1-Ad5-AL was effective for enhancing the inhibition effect and suppressing the migration of gastric cancer vascular endothelial cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development., (Published by Elsevier Ltd.)

Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 "safe harbor" locus on human chromosome 19., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Peste des petits ruminants is a highly contagious disease of small ruminants caused by a Morbillivirus, peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenovirus serotype 5 (Ad5) containing the highly immunogenic fusion protein (F) and hemaglutinine protein (H) genes from PPRV were constructed. HEK293A cells infected with either virus (Ad5-PPRV-F or -H) express F and H proteins respectively. These viruses were used to vaccinate mice by intramuscular inoculation. Both viruses elicited PPRV-specific B- and T-cell responses. Thus, after two immunizations, sera from immunized mice elicited neutralizing antibody response, indicating that this approach has the potential to confer protective immunity. In addition, we detected a significant antigen specific CD4(+) and CD8(+) T-cell response in mice vaccinated with either virus. These results indicate that these adenovirus constructs offer a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Autoimmune hepatitis type 2 (AIH-2) is a severe autoimmune liver disease with unknown etiology. We recently developed the CYP2D6 mouse model for AIH-2, in which mice are challenged with an adenovirus (Ad-2D6) expressing human cytochrome P450 2D6 (hCYP2D6), the major autoantigen in AIH-2. Such mice develop chronic hepatitis with cellular infiltrations and generation of hCYP2D6-specific antibodies and T cells. Importantly, the CYP2D6 model represents the only model displaying chronic fibrosis allowing for a detailed investigation of the mechanisms of chronic autoimmune-mediated liver fibrogenesis. We found that hCYP2D6-dependent chronic activation of hepatic stellate cells (HSC) resulted in an increased extracellular matrix deposition and elevated expression of α-smooth muscle actin predominantly in and underneath the liver capsule. The route of Ad-2D6 infection dramatically influenced the activation and trafficking of inflammatory monocytes, NK cells and hCYP2D6-specific T cells. Intraperitoneal Ad-2D6 infection caused subcapsular fibrosis and persistent clustering of inflammatory monocytes. In contrast, intravenous infection caused an accumulation of hCYP2D6-specific CD4 T cells throughout the liver parenchyma and induced a strong NK cell response preventing chronic HSC activation and fibrosis. In summary, we found that the location of the initial site of inflammation and autoantigen expression caused a differential cellular trafficking and activation and thereby determined the outcome of AIH-2-like hepatic damage and fibrosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Adenoviruses are used extensively to deliver genes into mammalian cells, particularly where there is a requirement for high-level expression of transgene products in cultured cells, or for use as recombinant viral vaccines or in gene therapy. In spite of their usefulness, the construction of adenoviral vectors (AdV) is a cumbersome and lengthy process that is not readily amenable to the generation of large collection of clones., Methods: In this project, to delete E1 gene in adenovirus, an adenoviral plasmid containing lateral sites of E1 region of adenovirus was made and recombination in the 293A cells between the homologous region of this linearized plasmid and the adenovirus genome resulted in the formation of the complete adenoviral recombinant., Results: This recombination resulted in loss of E1 region and we constructed a recombinant adenovirus type 5 vector that E1 gene was deleted by homologous recombination., Conclusion: Homologous recombination is more easy and fast technique in the production of AdV.

Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clinical trials. In the present review, the characteristics of the 'ideal' viral vector are discussed and the major types of viral vectors used in vascular gene transfer are reviewed. Basic knowledge of the use of viral vectors for direct in vivo gene transfer (adenoviral-based vectors, etc) and for ex vivo gene transfer (retroviral-based vectors) is provided. New developments in the field of viral vectorology, such as pseudotyping of retroviral vectors and targeting of other viral vectors to a specific cell type, will enhance the more rapid transition of vascular gene therapy from the experimental arena to the clinical setting.