Over the past several years, viral and non-viral gene delivery systems have been intensively developed to establish an ideal delivery vector for cancer gene therapy. Among the large variety of virus vectors currently being developed, the vector based on recombinant AAV (rAAV) is one of those that are closest to the ideal vector due to the following features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very limited host immune response and long-term persistent expression. However, there is concern that two major obstacles of AAV serotype 2 (AAV2), the relative paucity of AAV receptors on certain cell types and the presence of pre-existing immunity to AAV2 in humans might limit its clinical applications in humans. To overcome these limitations, we first investigated in vitro transduction efficiencies for 7 different AAV serotypes (AAV1 – AAV6, AAV8) in 12 human tumor cell lines (MKN74, AGS, NCI-H460, HT1080, HepG2, SK-Hep1, HCT-116, 5637, UMUC3, HeLa, DU145 and MCF7). We employed "pseudotyped" AAV vector systems, in which rAAV2 vector genomes containing the CMV promoter/ enhancer, the green fluorescent protein (GFP) cDNA and SV40 poly (A) signal, flanked by AAV2 ITR sequences are cross-packaged into the capsid proteins of the other AAV serotypes (several plasmids are kindly provided by Dr. Katherine A. High, University of Pennsylvania Medical Center). As we reported previously, the efficiency of rAAV2 infection into most of the tested cancer cell lines was significantly high (>90%), whereas the transduction efficiencies for other 5 different rAAV serotypes were varied and greatly low. On the other hand the efficiency of rAAV5 transduction in various tumor cell lines were comparably high, mostly similar to that of rAAV2. Using the xenograft nude mice implanted with either NCI-H460 or HCT-116 cancer cells, we also studied in vivo transduction efficiency for several pseudotyped rAAV vectors, which were shown to have a host range different from rAAV2. Taken altogether our results suggest that the pseudotyped rAAV5 vector can be developed as one of the most efficient gene delivery systems for cancer gene therapy to overcome the anti-AAV2 immune response. (This study was supported by the grant from the Ministry of Science and Technology (M10534040005-05N3404-00511), Seoul, and the Chung-Buk Pioneering Bioindustry R&D Grant, Chung-Buk, Korea.)
Abstract Age-related macular degeneration (AMD) is a prevalent retinal disorder that leads to central vision loss, mainly due to chronic inflammation. Tumor necrosis factor-alpha (TNF-α) is a critical mediator of inflammatory responses within the retinal environment. This study has investigated TNF-α's influence on inflammatory cytokine production and endothelial barrier integrity in human microglial (HMC3) and endothelial (HUVEC) cells. We found that TNF-α significantly elevated the expression and secretion of interleukin-6 (IL-6) and interleukin-1β (IL-1β) in HMC3 cells and disrupted endothelial tight junctions in HUVECs, as evidenced by weakened ZO-1 staining and compromised barrier function. To mitigate these effects and further investigate the in vitro mechanism of actions in CRG-01’s in vivo therapeutic efficacy of anti-inflammation, we employed AAV2-shmTOR, CRG-01, as the candidate for therapeutic vector targeting the mammalian target of the rapamycin (mTOR) pathway. TNF-α-induced IL-6, IL-1β, and NF-κB signaling in HMC3 cells were significantly reduced by AAV2-shmTOR treatment, which may present a promising avenue for the fight against AMD. It also effectively preserved endothelial tight junction integrity in TNF-α-treated HUVECs, providing reassurance about its effectiveness. Furthermore, the supernatant medium collected from AAV2-shmTOR-treated HMC3 cells decreased oxidative stress, protein oxidation, and cytotoxicity in ARPE retinal pigment epithelial cells. These results strongly suggested that CRG-01, the candidate therapeutic vector of AAV2-shmTOR, may have a therapeutic potential to treat AMD-related retinal inflammation.
s: Abstracts for the 20th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer (Primary Authors are Italicized): ANGIOGENESIS
Gene therapy has offered highly possible promises for treatment of cancers, as many potential therapeutic genes involved in regulation of molecular processes may be introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion, metastasis, and/or can stimulate the immune response against tumors. Therefore, viral and non-viral gene delivery systems have been developed to establish an ideal delivery vector for cancer gene therapy over the past several years. Among the currently developed virus vectors, the adeno-associated virus (AAV) vector is considered as one of those that are closest to the ideal vector mainly for genetic diseases due to the following prominent features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very low host immune response and long-term expression. Particularly, the most important attribute of AAV vectors is their safety profile in clinical trials ranging from CF to Parkinson's disease. Although adenovirus and several other oncolytic viruses have been more frequently used to develop cancer gene therapy, AAV also has many critical properties to be exploited for a cancer gene delivery vector. In this review, we will briefly summarize the basic biology of AAV and then mainly focus on recent progresses on AAV vector development and AAV-mediated therapeutic vectors for cancer gene therapy.
Abstract T-cell factor (TCF)-1 protein forms the transcriptional complex with β-catenin and regulates the expression of diverse target genes during early development and carcinogenesis. We have selected previously an RNA aptamer that binds to the DNA-binding domain of TCF-1 and have shown that it interfered with binding of TCF-1 to its specific DNA recognition sequences in vitro. As an approach to modulate the transcription by TCF/β-catenin complex in the cells, we have developed the RNA expression vector for stable expression of RNA aptamer inside of the mammalian cells. High level of RNA was expressed as an intramer in the fusion with the stable RNA transcript. The RNA intramer inhibited TCF/β-catenin transcription activity as shown by luciferase assay. It also modulated the expression of TCF/β-catenin target genes, such as cyclin D1 and matrix metalloproteinase-7, as predicted to be as an effective inhibitor of the TCF function. In addition, it efficiently reduced the growth rate and tumorigenic potential of HCT116 colon cancer cells. Such RNA intramer could lead to valuable gene therapeutics for TCF/β-catenin-mediated carcinogenesis. [Mol Cancer Ther 2006;5(9):2428–34]
To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound-healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.
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