• BMB Reports
• /
• v.53 no.11
• /
• pp.565-575
• /
• 2020

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replication-competent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.4
• /
• pp.1471-1476
• /
• 2012

Background and Aim: Currently available systemic therapies for malignant melanoma produce low response rates in patients, and more effective treatment modalities are clearly needed. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand has a significant impact on therapy for patients with X-linked inhibitor of apoptosis protein-downregulation malignant melanoma. The primary objective of this study was to assess its therapeutic potential. Materials and Methods: We employed a conditionally replicating oncolytic adenoviral vector, named CRAd5.TRAIL/siXIAP, with the characteristics of over-expression of the therapeutic gene TRAIL and downregulation of XIAP in one vector. B16F10-luc cells were employed to detect anti-tumor activity of CRAd5.TRAIL/siXIAP in vitro and in vivo. Results: CRAd5.TRAIL/siXIAP enhanced caspase-8 activation and caspase-3 maturation in B16F10 cells in vitro. Furthermore, it more effectively infected and killed melanoma cells in vitro and in vivo than other adenoviruses. Conclusion: Taken together, the combination of upregulation of TRAIL and downregulation of siXIAP with one oncolytic adenoviral vector holds promise for development of an effective therapy for melanomas and other common cancers.

• Korean Journal of Veterinary Research
• /
• v.51 no.3
• /
• pp.209-216
• /
• 2011

Four strains of fowl adenovirus (FAdV) were isolated from 4 flocks of broiler or layer chickens affected by hydropericardium syndrome in Korea. These FAdVs were classified as serotype 4 by restriction fragment length polymorphism patterns of hexon genes and whole genomes. The virus exhibited cytopathic effects consisting of rounding, ballooning and clustering in primary chicken embryo liver cell cultures. In transmission electron microscopy, virus particles in hexagonal shape aggregated exclusively in the nuclei of hepatocytes of the chickens as the typical appearances in adenovirus infections. Buoyant density of the virus in cesium chloride (CsCl) was 1.34 g/mL. The virus was stable to chloroform, ether, 50~70% ethanol, acidic condition at pH 3, 0.25% trypsin (1 : 250), heat at $50^{\circ}C$ for 30 min, but labile to 100% ethanol, heat at $52{\sim}60^{\circ}C$ for 30 min, 1 M $MgCl_2$ at $50^{\circ}C$ for 1 h, 1 : 2,000 formalin (37%). All of the physicochemical properties pertained to the characteristics of adenoviruses. Eight viral polypeptides were determined in CsCl-purified virus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• Molecules and Cells
• /
• v.40 no.8
• /
• pp.598-605
• /
• 2017

Human mesenchymal stem cells (MSCs) are currently being evaluated as a cell-based therapy for tissue injury and degenerative diseases. Recently, several methods have been suggested to further enhance the therapeutic functions of MSCs, including genetic modifications with tissue- and/or diseasespecific genes. The objective of this study was to examine the efficiency and stability of transduction using an adenoviral vector in human MSCs. Additionally, we aimed to assess the effects of transduction on the proliferation and multipotency of MSCs. The results indicate that MSCs can be transduced by adenoviruses in vitro, but high viral titers are necessary to achieve high efficiency. In addition, transduction at a higher multiplicity of infection (MOI) was associated with attenuated proliferation and senescence-like morphology. Furthermore, transduced MSCs showed a diminished capacity for adipogenic differentiation while retaining their potential to differentiate into osteocytes and chondrocytes. This work could contribute significantly to clinical trials of MSCs modified with therapeutic genes.

• Journal of Microbiology
• /
• v.41 no.4
• /
• pp.295-299
• /
• 2003

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.6
• /
• pp.2447-2451
• /
• 2014

Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

• BMB Reports
• /
• v.44 no.7
• /
• pp.446-451
• /
• 2011

The conversion of fibroblasts into osteoblasts requires the activation of key signaling pathways, including the BMP pathway. Although Runx2 is known to be a component of the BMP pathway, the combination of Runx2 and BMP2 has not yet been examined with respect to the conversion of fibroblasts into osteoblasts. Here, human ligamentum flavum (LF) fibroblast-like cells from six patients were tested for their conversion into osteoblasts using adenoviruses expressing Runx2 or BMP2. The forced expression of Runx2 or BMP2 in primary cultured LF cells resulted in a variety of proliferation and differentiation behaviors. Combined treatment of BMP2 plus Runx2 resulted in better osteoblastic differentiation than treatment with either component alone. These results indicate that the Runx2 and BMP2 pathways possess both common and independent target genes. Collectively, Runx2 plus BMP2 mediated efficient conversion of fibroblast-like LF cells into osteoblast-like cells, suggesting the possible use of these components for clinical applications such as spinal fusion.

• Pediatric Infection and Vaccine
• /
• v.3 no.1
• /
• pp.76-85
• /
• 1996

Adenoviruses(Ad) are considered to be second only to rotaviruses as the most significant cause of gastroenteritis in young children in Korea and thus it is essential to know the full spectrum of Ad serotypes routinely present in stool specimens from symptomatic patients. Sixty-six Ad isolates and three questionable ones collected over a 2-year peiord were typed by standard microneutralization, restriction endonuclease digestion and PCR of viral DNA to be able to evaluate these assays comprehensively for their ability to identify Ad associated with gastroenteritis. A total of sixty-one isolates(88.4%) were typed: the predominant types were Ad type 41(Ad41)(26.2%), Ad2(19.7%), Ad40(14.8%), Ad5(9.8%), and Ad7(9.8%) which together accounted for almost 80% of the isolates. The remaining virus isolates were typed as Ad1, 31, 34, 3, 25 and a mixture of 40/41. The incidence of Ad31(4.9%) or Ad3(1.6%) was relatively insignificant. DNA restriction analysis(77.5%) proved to be better than serum neutralization but not so when compared to a PCR-based assay for identification of the enteric Ad serotypes(90%) in stool specimens. In this work, the PCR-based assay was evaluated as a tool for the rapid, yet highly sensitive identification of adenoviral DNA sequences in fresh clinical stool specimens.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.3
• /
• pp.781-784
• /
• 2012

The aim of this study was to evaluate the effect of the adenovirus-mediated double suicide gene (CD/TK) for selective killing of gastric cancer cells. Gastric cancer cells SCG7901 and normal gastric epithelial cell lines were infected by adenoviruses Ad-survivin/GFP and Ad-survivin/CD/TK. GFP expression and CD-TK were detected by fluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. After treatment of the infected cells with the pro-drugs ganciclovir (GCV) and/or 5-FC, the cell growth status was evaluated by methyl thiazolyl tetrazolium assay. Cell cycle changes were detected using flow cytometry. In nude mice bearing human gastric cancer, the recombinant adenovirus vector was injected directly into the tumor followed by an intraperitoneal injection of GCV and/or 5-FC. The subsequent tumor growth was then observed. The GFP gene driven by survivin could be expressed within the gastric cancer line SCG7901, but not in normal gastric epithelial cells. RT-PCR demonstrated the presence of the CD/TK gene product in the infected SCG7901 cells, but not in the infected normal gastric epithelial cells. The infected gastric cancer SCG7901, but not the gastric cells, was highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide genes in killing the target cells (P<0.01). Treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G0-Gl phase and decreased percentage in S phase. In nude mice bearing SCG7901 cells, treatment with the double suicide gene system significantly inhibited tumor growth, showing much stronger effects than either of the single suicide genes (P<0.01). The adenovirus-mediated CD/TK double suicide gene driven by survivin promoter combined with GCV an 5-FC treatment could be an effective therapy against experimental gastric cancer with much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.

• Molecular & Cellular Toxicology
• /
• v.3 no.2
• /
• pp.85-89
• /
• 2007

Efficient gene delivery to specific tissues, such as inflammatory and cancerous tissues, is currently a major concern in disease treatment. The human transferrin receptor (hTR) has been detected in the synovium and fibroblast-like synoviocytes (FLS), which raises the possibility that expression of hTR is associated with the pathogenesis of rheumatoid arthritis (RA). To investigate whether the hTR is a useful target for gene transduction into the FLS of RA patients, recombinant adenoviruses with wildtype fiber (AdLac) and transferrin peptide-tagged fiber (Tf-AdLac) were used. The hTR expression level in FLS was notably increased by basic fibroblast growth factor (bFGF). Gene transduction to FLS was significantly higher by the hTR-targeted adenovirus than by the AdLac adenovirus, and treatment of the FLS with bFGF resulted in increased gene transduction by Tf-AdLac. Taken together, these data support Tf-AdLac as a new strategy for gene transduction in the treatment of RA patients.