Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs

PLoS One. 2016 Oct 20;11(10):e0164720. doi: 10.1371/journal.pone.0164720. eCollection 2016.

Abstract

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.

MeSH terms

  • Cells, Cultured
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression Regulation, Viral
  • Genetic Vectors
  • HeLa Cells
  • Humans
  • MicroRNAs / genetics*
  • Organ Specificity
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism
  • Protein C / genetics
  • Protein C / metabolism*
  • Sendai virus / genetics*
  • Transduction, Genetic
  • Transgenes*

Substances

  • MIRN21 microRNA, human
  • MicroRNAs
  • Protein C
  • mirnlet7 microRNA, human

Grant support

This work was supported in part by JSPS KAKENHI Grant Number 24700490 (to MS) and by Program for Creating STart-ups from Advanced Research and Technology (START) (Japan Science and Technology Agency) (to MN). MN received support in the form of salary as founder and CTO (Chief Technology Officer) at Tokiwa-Bio. Inc. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.