Abstract
RNA interference (RNAi) has emerged as a promising technique for gene therapy. However, the safe and long-term expression of small RNA molecules is a major concern for the application of RNAi therapies in vivo. Borna disease virus (BDV), a non-segmented, negative-strand RNA virus, establishes a persistent infection without obvious cytopathic effects. Unique among animal non-retroviral RNA viruses, BDV persistently establishes a long-lasting persistent infection in the nucleus. These features make BDV ideal for RNA virus vector persistently expressing small RNAs. Here, we demonstrated that the recombinant BDV (rBDV) containing the miR-155 precursor, rBDV-miR-155, persistently expressed miR-155 and efficiently silenced its target gene. The stem region of the miR-155 precursor in rBDV-miR-155 was replaceable by any miRNA sequences of interest and that such rBDVs efficiently silence the expression of target genes. Collectively, BDV vector would be a novel RNA virus vector enabling the long-term expression of miRNAs for RNAi therapies.
Highlights
Small RNA molecules, such as small interfering RNAs, microRNAs and short-hairpin RNAs, are central to RNA interference (RNAi), a naturally occurring regulatory mechanism causing posttranscriptional gene silencing in most eukaryotic cells1,2. siRNAs are short (21- to 25-nucleotides [nt]) dsRNAs that mediate mRNA degradation in a sequence-specific manner[3]
Vectors based on negative-strand RNA viruses (NSV) for expression of small RNAs have been reported rarely because almost all RNA viruses are transcribed in the cytoplasm, whereas the processing machinery of pri-miRNAs is in the nucleus
To ensure that virus-derived miR-155 was produced by the endogenous miRNA machinery, we examined the gene silencing of recombinant BDV (rBDV)-miR-155 in the presence of siRNA against Dicer
Summary
Small RNA molecules, such as small interfering RNAs (siRNA), microRNAs (miRNA) and short-hairpin RNAs (shRNA), are central to RNA interference (RNAi), a naturally occurring regulatory mechanism causing posttranscriptional gene silencing in most eukaryotic cells1,2. siRNAs are short (21- to 25-nucleotides [nt]) dsRNAs that mediate mRNA degradation in a sequence-specific manner[3]. SiRNAs can be introduced into cells as synthetic oligonucleotides, vector-derived expression of miRNAs would be more useful for the efficient and persistent expression of small RNA molecules because a vector-derived transcript can be processed into multiple, functional miRNAs. Currently, viral vector systems are used widely for the expression of miRNAs and shRNAs. Adeno-associated virus (AAV), adenovirus and retrovirus vectors are commonly used as delivery systems for these small RNA molecules. We established a novel RNA virus-based vector using Borna disease virus (BDV) reverse genetics[10,11] This BDV vector harbors an extra transcription cassette in the intercistronic non-coding region between the viral phosphoprotein (P) and matrix (M) genes and can express foreign genes efficiently and stably in infected cells. These characteristics make BDV the only animal non-retroviral RNA virus capable of intranuclear parasitism, offering the possibility that it may be an ideal candidate for the long-term expression system of small RNA
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