Abstract
Viral vectors are efficient gene delivery systems, although most of these vectors still present limitations to their practical use, such as achieving only transient transgene expression and a risk of insertional mutations. We have recently developed an RNA virus-based episomal vector (REVec), based on nuclear-replicating Borna disease virus (BoDV). REVec can transduce transgenes into various types of cells and stably express transgenes; however, an obstacle to the practical use of REVec is the lack of a mechanism to turn off transgene expression once REVec is transduced. Here, we developed a novel REVec system, REVec-L2b9, in which transgene expression can be switched on and off by using a theophylline-dependent self-cleaving riboswitch. Transgene expression from REVec-L2b9 was suppressed in the absence of theophylline and induced by theophylline administration. Conversely, transgene expression from REVec-L2b9 was switched off by removing theophylline. To our knowledge, REVec-L2b9 is the first nuclear-replicating RNA virus vector capable of switching transgene expression on and off as needed, which will expand the potential for gene therapies by increasing safety and usability.
Highlights
Increasing lists of causative genes for genetic disorders and gene delivery systems have led to the development of gene therapies over several decades
We have recently developed an RNA virus-based episomal vector (REVec), based on nuclear-replicating Borna disease virus (BoDV)
REVec-Gaussia Luciferase (GLuc)-L2b9s contained the L2b9 ribozyme sequence in the REVec antigenomic RNAs, the replication kinetics of REVec-GLuc-L2b9s were comparable to those of REVec-GFP and REVec-GLuc during the de novo infection (Figure 1F). These results indicate that the insertion of the L2b9 sequence in REVec antigenomic RNA does not induce any deleterious effects on vector replication in the infected cells
Summary
Increasing lists of causative genes for genetic disorders and gene delivery systems have led to the development of gene therapies over several decades. Various non-viral gene delivery systems, such as cationic liposome technology (Miller, 1998), and viral delivery systems, such as adeno virus-, adeno-associated virus (AAV)-, and lentivirusbased vectors, have been developed (Escors and Breckpot, 2010; Crystal, 2014; Naso et al, 2017). These systems can be used to successfully transduce a gene of interest, they still present disadvantages such as achieving only transient expression of transgenes, cytotoxicity, and the potential for integration of viral vector sequences into the host genome. Possible requirements for an ideal gene delivery system include longterm expression of transgenes, minimal cytotoxicity, reduction of integration risk, and controllable
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