Abstract
The adenovirus (Ad) genome encodes one or two non-coding small RNAs called virus-associated (VA)-RNAs, that are transcribed by polymerase III and support Ad replication. As previously reported, a replication-incompetent Ad vector, which is widely used in not only gene therapy studies, including clinical trials, but also basic researches as a gene delivery vehicle, as well as wild-type Ad (WT-Ad) express VA-RNAs, and VA-RNAs activate innate immunity, including the production of type I interferons. In addition, VA-RNAs perturb cellular microRNA (miRNA) expression profiles via competitive inhibition of key components involved in the miRNA maturation pathway. Although these characteristics of VA-RNAs might negatively affect the application of Ad vectors, VA-RNA expression profiles following transduction with an Ad vector have been not fully examined. In this study, we quantitatively analyzed the expression profiles of VA-RNAI, which is a major species of VA-RNAs, following transduction with Ad vectors in vitro and in vivo using real-time RT-PCR. The VA-RNAI expression levels in the cells transduced with a conventional Ad vector expressing luciferase (Ad-CAL2) at a multiplicity of infection (MOI) of 100 were approximately 2000- to 3000-fold lower than those infected with WT-Ad at the same MOI at 48 h after treatment. The expression levels of VA-RNAI in the mouse liver following administration with Ad-CAL2 were approximately 600-fold lower than those following administration with WT-Ad at 48 h post-administration. miRNA-mediated suppression of leaky expression of the Ad E4 genes resulted in about five-fold reduction in the VA-RNAI copy numbers in the liver following systemic administration in mice. These data provide informative clues for the development of novel safer Ad vectors.
Highlights
A replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy as well as basic research thanks to several advantages as a gene delivery vehicle, such as hightiter production and highly efficient transduction into a variety of dividing and non-dividing cells in vitro and in vivo
We and Minamitani et al [10] demonstrated that a conventional replication-incompetent Ad vector as well as wild-type Ad (WT-Ad) each express two small RNAs, which are approximately 160nt-long non-coding RNAs transcribed by RNA polymerase III, and that VA-RNAs induce innate immune responses via the retinoic acid-inducible gene I (RIGI) and/or interferon (IFN)-beta promoter stimulator-1 (IPS-1) pathway [8,10]
In order to examine the expression profiles of VA-RNAI in cultured cells following transduction with an Ad vector, HUVEC and SK HEP-1 cells were transduced with Ad-CAL2, a replicationincompetent Ad vector expressing luciferase, at multiplicity of infection (MOI) of 10 and 100
Summary
A replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy as well as basic research thanks to several advantages as a gene delivery vehicle, such as hightiter production and highly efficient transduction into a variety of dividing and non-dividing cells in vitro and in vivo. It is well known that systemic administration of Ad vectors causes innate and adaptive immune responses, leading to multi-tissue damage, including hepatotoxicity [1,2]. VA-RNAs inhibit microRNA (miRNA) production by competitive inhibition of key components involved in the miRNA processing pathway, such as exportin-5, dicer, and argonaute 2 [11,12]. These characteristics of VA-RNAs might contribute to Ad vector-induced hepatotoxicities, which are main side effects of systemic administration of an Ad vector, because inhibition of miRNA production in the liver leads to severe hepatotoxicities [13]
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