Search

Nonhuman adenoviruses including bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector gene expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as an alternative or supplement to HAd5 for gene delivery applications.

Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH) in liver resulting in blood phenylalanine (Phe) elevation and neurotoxicity. A pegylated phenylalanine ammonia lyase (PEG-PAL) metabolizing Phe into cinnamic acid was recently approved as treatment for PKU patients. A potentially one-time rAAV-based delivery of PAH gene into liver to convert Phe into tyrosine (Tyr), a normal way of Phe metabolism, has now also entered the clinic. To understand differences between these two Phe lowering strategies, we evaluated PAH and PAL expression in livers of PAHenu2 mice on brain and liver functions. Both lowered brain Phe and increased neurotransmitter levels and corrected animal behavior. However, PAL delivery required dose optimization, did not elevate brain Tyr levels and resulted in an immune response. The effect of hyperphenylalanemia on liver functions in PKU mice was assessed by transcriptome and proteomic analyses. We observed an elevation in Cyp4a10/14 proteins involved in lipid metabolism and upregulation of genes involved in cholesterol biosynthesis. Majority of the gene expression changes were corrected by PAH and PAL delivery though the role of these changes in PKU pathology is currently unclear. Taken together, here we show that blood Phe lowering strategy using PAH or PAL corrects both brain pathology as well as previously unknown lipid metabolism associated pathway changes in liver. [ABSTRACT FROM AUTHOR]

Abstract: Nonhuman adenovirus (Ad) vectors derived from bovine Ad serotype 3 (BAd3) or porcine Ad serotype 3 (PAd3) can circumvent pre-existing immunity against human Ad (HAd). We have previously reported differential transduction of human and nonhuman cells by these Ad vectors, and their distinct receptor usage and biodistribution. To compare the induction of innate immunity, vector toxicity and vector uptake by Kupffer cells (KCs) following intravenous administration of PAd3, BAd3, or HAd5 vectors in mice, we determined mRNA expression levels of proinflammatory chemokines and cytokines, and Toll-like receptors (TLRs) in the liver and spleen. Tissue toxicity of these vectors was assessed by comparing serum levels of liver-specific enzymes, histopathology and Kupffer cell (KC) depletion. Compared to the HAd5 vector, PAd3 and BAd3 vectors were more potent stimulators of innate immune responses as indicated by enhanced mRNA expression of TLRs and proinflammatory chemokines and cytokine genes. Histopathological changes in the liver were most pronounced in HAd5-inoculated mice while BAd3- or PAd3-inoculated mice revealed mild histologic changes that were confined to early time points. Inoculation with HAd5 or PAd3 vectors resulted in a significant (P <0.05) decline of the number of KCs in the liver. Together, these results extend our previous observations regarding distinct in vivo biology of nonhuman and human Ad vectors. [Copyright &y& Elsevier]

The EphA2 receptor tyrosine kinase is frequently overexpressed in invasive breast cancer cells. Moreover, these malignant cells have unstable cell-cell contacts, which preclude EphA2 from interacting with its ligand, EphrinA1, which is anchored to the membrane of adjacent cells. This defect is important because ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and survival, whereas the absence of ligand binding favors these same behaviors. In our present study, human adenoviral type 5 (HAd) vectors were engineered to express secreted-forms of EphrinA1. These vectors were used to infect MDA-MB-231 human breast cancer cells, or MCF-10A human breast epithelial cells providing matched controls. Infection with HAd-EphrinA1-Fc (HAd vector expressing extracellular domain of human EphrinA1 attached to Fc portion of human IgG1 heavy chain) caused increased EphA2 activation and turnover and consequently decreased tumor cell viability in soft agar assays. Consistent with this observation, infection of MDA-MB-231 cells with HAd-EphrinA1-Fc prevented tumor formation in xenograft models. Furthermore, therapeutic modeling via intratumoral inoculation revealed that HAd-EphrinA1-Fc significantly inhibited subsequent tumor growth as compared to matched controls. These results suggest that targeting of EphA2 with adenoviral vectors may have therapeutic value.Cancer Gene Therapy (2004) 11, 757-766. doi:10.1038/sj.cgt.7700761 Published online 10 September 2004 [ABSTRACT FROM AUTHOR]

Nonhuman adenoviral (Ad) vectors have shown the potential to overcome the preexisting Ad immunity. In order to assess their utility for in vivo applications, it is important to evaluate biodistribution, induction of innate immune response, and toxicity of these vectors in a suitable animal model. In this study, FVB/n mice were inoculated via the intravenous route with porcine Ad type 3 (PAd3), bovine Ad type 3 (BAd3), or human Ad type 5 (HAd5) vector carrying the green fluorescent protein (GFP) gene as a reporter. Mice inoculated similarly with phosphate-buffered saline served as negative controls. Mice were sacrificed at 0.25, 0.5, 1, 2, 4, 8 and 16 days post-inoculation, and the blood, liver, spleen, kidneys, lungs and heart were collected for further analyses. Vector biodistribution and transgene expression were analyzed by real-time PCR and reverse-transcription PCR, respectively. The serum samples were analyzed for induction of inflammatory cytokines such as tumor necrosis factor (TNF) alpha (TNF-a), interleukin 6 (IL-6), and interferon gamma (IFN-g). Hepatotoxicity of Ad vectors was assessed by monitoring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and also by histopathology. At early time points (up to 2 days post-inoculation), the presence of PAd3 vector was detected in the lungs, spleen, and liver, and the BAd3 vector was detected in all tissues examined. Whereas, the presence of HAd5 vector was detected only in the liver suggesting that biodistribution of these nonhuman Ad vectors was considerably different than that of HAd5. Increased serum IL-6 levels were observed at 0.25 day post-inoculation in the PAd3-inoculated group, while increased IFN-g levels were seen on 8 days post-inoculation in the HAd5-inoculated group. Serum AST and ALT levels were elevated at early time points (up to 1day post-inoculation) in mice inoculated with either PAd3 or BAd3, but not in the HAd5-inoculated group. Surprisingly, elevated levels of these enzymes were noted at a late time point (8 days post-inoculation) only in the HAd5-inoculated group. Histopathologically, the PAd3 vector elicited mild hepatocellular apoptosis, and the HAd5 vector resulted in mild multifocal lymphocytic hepatitis, but no hepatotoxicity was observed in the BAd3-inoculated group. These results indicated that biodistribution, innate immune response and hepatotoxicity following intravenous inoculation of mice with PAd3, BAd3, or HAd5 vector were different. Further characterization of nonhuman Ad vectors will be valuable for designing improved Ad-based gene delivery vectors.Molecular Therapy (2006) 13, S7–S7; doi: 10.1016/j.ymthe.2006.08.028 [ABSTRACT FROM AUTHOR]

Abstract: The potential of a bovine adenovirus serotype 3 (BAd3)-based vector to bypass the human adenoviral serotype 5 (HAd5)-specific neutralizing immune response was evaluated in an immunocompetent mouse model of breast cancer. Initially we monitored vector biodistribution, genome persistence, transgene expression, and potential toxicity of HAd-GFP [HAd5 vector expressing green fluorescent protein (GFP)] or BAd-GFP (BAd3 vector expressing GFP) in FVB/n mice bearing tumors. A comparable biodistribution pattern for BAd-GFP and HAd-GFP was evident. In addition, following the development of vector-specific immune responses, animals were inoculated intratumorally (i.t.) with HAd-GFP or BAd-GFP. HAd-GFP immunity did not hamper the transduction and persistence of BAd-GFP into the tumors and other organs, and, similarly, BAd-GFP immunity did not hamper the transduction and persistence of HAd-GFP. Both BAd3 and HAd5 vectors showed relatively higher transgene expression in the presence of heterologous vector immunity. In contrast, the homologous vector immunity was associated with a rapid vector clearance and decline in transgene expression levels. Histopathological changes in BAd-GFP inoculated animals were generally mild with some acute but recoverable hepatic perturbations. Overall, the data suggest the importance of BAd3 vectors for sequential vector administration in overcoming the vector immunity for cancer gene therapy. [Copyright &y& Elsevier]