Species D human adenovirus type 9 exhibits better virus-spread ability for antitumor efficacy among alternative serotypes

Species C human adenovirus serotype 5 (HAdV-C5) is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR), HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR)-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5

A multi targeting conditionally replicating adenovirus displays enhanced oncolysis while maintaining expression of immunotherapeutic agents

Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis

Therapeutic efficacy of an oncolytic adenovirus containing RGD ligand in minor capsid protein IX and Fiber, Δ24DoubleRGD, in an ovarian cancer model

Ovarian cancer is the leading cause of gynecological disease death despite advances in medicine. Therefore, novel strategies are required for ovarian cancer therapy. Conditionally replicative adenoviruses (CRAds), genetically modified as anti-cancer therapeutics, are one of the most attractive candidate agents for cancer therapy. However, a paucity of coxsackie B virus and adenovirus receptor (CAR) expression on the surface of ovarian cancer cells has impeded treatment of ovarian cancer using this approach.This study sought to engineer a CRAd with enhanced oncolytic ability in ovarian cancer cells, “Δ24DoubleRGD.” Δ24DoubleRGD carries an arginine-glycine-aspartate (RGD) motif incorporated into both fiber and capsid protein IX (pIX) and its oncolytic efficacy was evaluated in ovarian cancer. In vitro analysis of cell viability showed that infection of ovarian cancer cells with Δ24DoubleRGD leads to increased cell killing relative to the control CRAds. Data from this study suggested that not only an increase in number of RGD motifs on the CRAd capsid, but also a change in the repertoir of targeted integrins could lead to enhanced oncolytic potency of Δ24DoubleRGD in ovarian cancer cells in vitro. In an intraperitoneal model of ovarian cancer, mice injected with Δ24DoubleRGD showed, however, a similar survival rate as mice treated with control CRAds

Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy

<p>Abstract</p> <p>Background</p> <p>Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTD_tat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTD_tatmotif would improve the efficacy of Ad5-mediated gene delivery.</p> <p>Results</p> <p>In this study, we genetically incorporated the PTD_tatmotif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTD_tat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTD_tatexhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTD_tatwas mediated by binding of the positively charged PTD_tatpeptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated <it>in vivo </it>gene delivery efficacy of Ad5.PTD_tatusing subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTD_tatexhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique.</p> <p>Conclusion</p> <p>Genetic incorporation of the PTD_tatmotif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTD_tattargeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in <it>in vivo </it>tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications.</p

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-211The nucleotide sequence reported herein has been deposited in the DDBJ, EMBL and GenBank databanks under the accession number AB034697.

AbstractA mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription

Evaluation of adenovirus capsid labeling versus transgene expression

Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout

Derivation of a Triple Mosaic Adenovirus for Cancer Gene Therapy

A safe and efficacious cancer medicine is necessary due to the increasing population of cancer patients whose particular diseases cannot be cured by the currently available treatment. Adenoviral (Ad) vectors represent a promising therapeutic medicine for human cancer therapy. However, several improvements are needed in order for Ad vectors to be effective cancer therapeutics, which include, but are not limited to, improvement of cellular uptake, enhanced cancer cell killing activity, and the capability of vector visualization and tracking once injected into the patients. To this end, we attempted to develop an Ad as a multifunctional platform incorporating targeting, imaging, and therapeutic motifs. In this study, we explored the utility of this proposed platform by generating an Ad vector containing the poly-lysine (pK), the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and the monomeric red fluorescent protein (mRFP1) as targeting, tumor cell killing, and imaging motifs, respectively. Our study herein demonstrates the generation of the triple mosaic Ad vector with pK, HSV-1 TK, and mRFP1 at the carboxyl termini of Ad minor capsid protein IX (pIX). In addition, the functionalities of pK, HSV-1 TK, and mRFP1 proteins on the Ad vector were retained as confirmed by corresponding functional assays, indicating the potential multifunctional application of this new Ad vector for cancer gene therapy. The validation of the triple mosaic Ad vectors also argues for the ability of pIX modification as a base for the development of multifunctional Ad vectors

Cellular uptake of HAdV-D9 independently of human CAR.

<p>Cells (CHO and CHO-hCAR) were infected with HAdV-C5 or HAdV-D9 at an MOI of 100 genomes/cell, and total cell DNA extracted from infected cells were used for qPCR analysis. (A) Cellular uptakes of HAdVs in CHO and CHO-hCAR were represented as a fold of genome transfer in HAdV-C5-infected CHO cells at 1 hour post-infection and normalized by cellular uptake of HAdV-C5 in CHO cells; HAdV-C5 (black bars) and HAdV-D9 (white bars). (B and C) Cellular uptakes of HAdVs in a time-dependent manner in CHO (B) and CHO-hCAR (C). Total cell DNA was extracted from infected cells at 0, 30, and 60 min post-infection and used for qPCR analysis; HAdV-C5 (black squares) and HAdV-D9 (white squares). Data points represent mean+standard error of the mean (n = 3). CHO; human CAR negative CHO cells and CHO-hCAR; CHO cells stably expressing human CAR. (D) CHO-hCAR cells were treated with the HAdV-C5 fiber knob protein at a final concentration of 0, 0.5, 5.0 or 50 µg/ml at 4°C for 1 hour and then infected with HAdV at an MOI of 100 genomes/cell at 37°C for 1 hour. HAdV-C5 (black bars) and HAdV-D9 (white bars). (E) Cellular uptakes of HAdV-D9 in cancer cells which express little or no hCAR. Cells were infected with HAdV-C5 or HAdV-D9 at an MOI of 100 genomes/cell for 1 hour post-infection, and total cell DNA extracted from infected cells was analyzed by qPCR. HAdV-C5 (black bars) and HAdV-D9 (white bars). Cellular uptakes of HAdVs in cell lines were represented as (A) a fold of genome transfer, (B, C and D) a percentage of genome transfer, and (E) Genome copy numbers per 50 ng of total DNA of each HAdV. Data points represent mean+standard error of the mean (n = 3).</p

Plaque morphology of HAdVs on A549 cells.

<p>HAdVs (HAdV-C5, D9, D51, E4, and F41) were serially diluted with medium containing 2% FBS and A549 cells were infected with HAdV at the range of dilution of 5.0×10⁻⁸ and 5.0×10⁻⁹ for 1 hour. We performed plaque assay as described in the Materials and Methods section. At 14 days post-infection, we stained cells with 2 ml of medium containing 0.75% agar and 0.033% neutral red in order to visualize individual single plaques on A549 cells. Photographs of plaque morphology of HAdVs were taken with a digital camera.</p

Release of HAdV-D9 from infected cells to culture medium.

<p>A549 cells were infected with HAdV-C5 or HAdV-D9 at an MOI of 10 PFU/cell and harvested at various time points. Samples to measure the infectious titer of HAdVs were prepared from infected cells harvested along with culture medium (A), a fraction extracted from infected cells without culture medium (B), and culture medium (C). Infectious titers of HAdVs contained in each fraction were measured by triplicate TCID50 assays. Data points represent mean+standard error of the mean (n = 3). Unpaired student <i>t</i>-test analysis was performed with respect to HAdV-C5 at each time point and significance is indicated by *<i>P</i><0.05.</p