Efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery

Abstract

First-generation adenovirus vectors (FG AdVs) are widely used in basic studies and gene therapy. However, virus-associated (VA) RNAs that act as small-interference RNAs are indeed transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expressions of numerous cellular genes. Therefore, VA-deleted AdVs lacking VA RNA genes would be advantageous for basic studies, both in vitro and in vivo. Here, we describe an efficient method of producing VA-deleted AdVs. First, a VA RNA-substituted “pre-vector” lacking the original VA RNA genes but alternatively possessing an intact VA RNA region flanked by a pair of FRTs was constructed. VA-deleted AdVs were efficiently obtained by infecting 293hde12 cells, which highly express FLP, with the pre-vector. The resulting transduction titers of VA-deleted AdVs were sufficient for practical use. Therefore, VA-deleted AdVs may be substitute for current FG AdV.

Adenovirus VA RNA: An essential pro-viral non-coding RNA

“…Understanding the multiple ways in which VA RNA I affects cellular processes has been critical for the efficient development of AdV-based technologies for gene therapy, oncotherapy, and vaccine development (Machitani et al, 2011(Machitani et al, , 2013Maekawa et al, 2013). While the efficiency with which VA RNA I inhibits PKR has made this RNA a valuable tool for ensuring effective translation of transfected proteins (Promega), VA RNAs can be detrimental to experimental or therapeutic introduction of small hairpin RNA (shRNA) for gene silencing because of the way in which VA RNA interacts with Dicer and associates with the RISC complex .…”

“…In addition to acute infection, AdVs are capable of both persistent (Fox et al, 1977) and true latent infection (Garnett et al, 2009;Neumann et al, 1987). In spite of their complex biology, our depth of knowledge of AdV coupled with its ease of genetic manipulation, ability to infect a wide range of tissues, including both dividing and senescent cells, has lead to its development as a promising candidate for gene and oncotherapy (Choi et al, 2012;Maekawa et al, 2013).…”

Adenovirus-Encoding Virus-Associated RNAs Suppress HDGF Gene Expression to Support Efficient Viral Replication

“…The VA-deleted AdVs except for HDGF- and GFP-expressing AdVs were prepared according to a method using 293U6VA-1 cells that constitutively express both VAI and VAII. HDGF-expressing and GFP-expressing VA-deleted AdVs were generated as described previously [16] . Briefly, an HDGF-expressing and a GFP-expressing unit under the control of the EF1α promoter was inserted into the SwaI cloning site at the authentic E1 substitution region in the pre-vector cosmid pAxdV-4FVF-w, and the pre-vectors were prepared using 293 cells.…”

“…For this purpose, AdVs lacking VA RNA genes (VA-deleted AdVs) are essential as a control; however, VA-deleted AdVs have been difficult to generate and produce in quantities sufficient for practical use. Recently, we have developed a novel method for the efficient production of VA-deleted AdVs using a site-specific recombinase FLP [16] . A “pre-vector” containing the VA RNA region flanked by a pair of FRT sequences, which are target sequences for FLP recombinase, is constructed according to a commonly used method for the production of FG AdV [17] .…”

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

“…VA-deleted AdVs were prepared according to the method using 293hde12 cells described by Maekawa et al 12 or using 293U6VA-1 cells that constitutively express both VAI and VAII. The VA-deleted AdVs in this study grew efficiently in this cell line.…”