RNAi by short hairpin RNA (shRNA) is a powerful tool not only for studying gene functions in various organisms, including mammals, but also for the treatment of severe disorders. However, shRNA-expressing vectors can induce type I interferon (IFN) expression by activation of innate immune responses, leading to off-target effects and unexpected side effects. Several strategies have been developed to prevent type I IFN induction. On the other hand, it has remained unclear whether type I IFNs have effects on shRNA-mediated RNAi. Here, we show that the type I IFNs significantly inhibit shRNA-mediated RNAi. Treatment with recombinant human IFN-α significantly inhibited shRNA-mediated knockdown of target genes, while it did not inhibit small interfering RNA (siRNA)-mediated knockdown. Following treatment with IFN-α, increased and decreased copy numbers of shRNA and its processed form, respectively, were found in the cells transfected with shRNA-expressing plasmids. Dicer protein levels were not altered by IFN-α. These results indicate that type I IFNs inhibit shRNA-mediated RNAi via inhibition of dicer-mediated processing of shRNA to siRNA. Our findings should provide important clues for efficient RNAi-mediated knockdown of target genes in both basic researches and clinical gene therapy.
Background/Aim: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for selfreplication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd. Materials and Methods: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds. Results: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds. Conclusion: These results provide important clues for the development of safe and efficient OAds.Oncolytic virotherapies using oncolytic viruses, which replicate in a tumor cell-specific manner and mediate efficient tumor cell killing activities, have attracted much attention as a novel therapeutic approach for various types of tumors (1, 2). More than 10 species of oncolytic viruses, including adenovirus, herpes virus, and reovirus, have been developed. Pre-clinical and clinical trials using oncolytic viruses have been carried out worldwide and have shown promising results. Talimogene laherparepvec (T-VEC), a genetically modified herpes simplex virus type 1 (HSV-1), was approved by the U.S. Food and Drug Administration (FDA) in 2015 as a first-in-class oncolytic virus for the treatment of metastatic melanoma.Among the various types of oncolytic viruses, the most promising are the oncolytic adenoviruses (OAds); their several advantages include efficient tumor cell killing activities and relatively large capacities for transgene insertion (3). In OAds, the E1A gene is crucial for regulation of the virus gene expression and self-replication of the virus genome. This gene must function or be expressed in a tumor cell-specific manner in order to permit tumor cell-specific infection, making the design of the E1A gene expression cassette crucial for efficient and tumor cell-specific infection with oncolytic adenoviruses (OAds) without cytotoxicity to normal cells. Various types of E1A gene expression cassettes have been developed, but they can be largely divided into two groups. In the most widely used type of E1A gene expression cassette, the E1A gene is driven by a tumorspecific promoter, including human telomerase reverse transcriptase (hTERT) promoter and survivin promoter (4, 5). In the other type of cas...
Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer agents. Numerous studies have examined the antitumour effects of combinational use of an OAd and anticancer agents; however, few chemical compounds enhancing OAd infection have been reported. In this study, we screened a food and drug administration (FDA)-approved drug library containing 1134 small chemical compounds to identify chemical compounds that enhance OAd replication in human tumour cells. We found that domperidone, a dopamine D2 receptor antagonist, significantly enhanced the replication of an OAd in human tumour cells, including human pancreatic tumour cells, by two–fivefold, resulting in improvement of OAd-mediated tumour cell killing activities. The E1A mRNA levels were significantly increased in domperidone-pre-treated cells following OAd infection, which contributed to the promotion of OAd replication. However, mRNA levels of the dopamine D2 receptor (DRD2), which is known to be a target molecule of domperidone, were undetectable in most of the tumour cells by real-time reverse transcription (RT)-PCR analysis, indicating that domperidone promoted OAd replication by acting on a molecule other than DRD2. This study provides important clues for the improvement of OAd-mediated cancer therapy.
Background/Aim: Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies. Materials and Methods: We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors. Results: OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth. Conclusion: OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer.Pancreatic cancer is the fourth leading cause of cancerrelated death (1, 2). The 5-year survival rates are about 10% for all stages combined and 15-20% for surgically resected patients (3-6). Due to the lack of effective treatments without severe toxicity profiles, the 5-year survival rates of patients who cannot undergo surgical resection have remained unchanged (7,8). Since pancreatic cancer is often an aggressive malignancy and does not exhibit obvious symptoms, many patients present with unresectable disease or distant metastases at the time of diagnosis. The 5-year survival rate of patients with unresectable disease is less than 5%. Moreover, undetectable distant metastases make a major contribution to the low 5-year survival rate of patients undergoing surgical resection (9). Thus, there is an urgent need for the development of novel, systemically deliverable 537
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