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, 11 (10), e0164720
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Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs

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

Masayuki Sano et al. PLoS One.

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.

Conflict of interest statement

MN is a founder and CTO (Chief Technology Officer) at Tokiwa-Bio. Inc. There are no patents, products in development, or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Inhibitory effect of SeV C on transgene expression mediated by the SeVdp vector.
(A) Genome structure of SeV-EGFP. The NP, P, and L genes are indispensable for genome replication and transcription. The P gene contains multiple open reading frames encoding the P, C, and V proteins. Hygromycin resistance gene (Hygr) and EGFP genes were inserted into the SeVdp backbone as transgenes. (B) Inhibition of transgene expression by SeV C overexpression. The plasmid containing either the human codon-optimized C gene (pCMV-C) or puromycin resistance gene (pCMV-Pur) under control of the CMV enhancer/promoter was transfected into HeLa S3 cells harboring the SeV-EGFP, and the levels of EGFP expression were measured by flow cytometry. The EGFP intensity of cells treated with transfection reagent (mock) was set to 1.0, and relative fluorescence intensities are indicated. The means and standard deviations (SD) from three replicate experiments are presented.
Fig 2
Fig 2. Effect of SeVdp-mediated C overexpression on transgene expression.
(A) Genome structure of a series of SeVdp vectors. C-opt: human codon-optimized C gene, Puror: puromycin resistance gene. (B) Inhibition of transgene expression by SeVdp-mediated C overexpression. HeLa S3 cells were infected with different SeVdp vectors and treated with hygromycin B. The levels of EGFP expression were measured by flow cytometry. EGFP intensity in SeV-Pur cells was set to 1.0, and relative fluorescence intensities of infected cells are indicated. Non-infected cells (mock) were used as a negative control. The means and SD from three replicate experiments are presented. (C) Effect of siRNA against the C-opt gene on transgene expression mediated by SeVdp vectors. Negative control siRNA (siNEG) or siRNA against the C-opt gene (siC-opt) was transfected into SeV-Pur2 or SeV-C2 cells. The levels of EGFP expression were measured by flow cytometry. The EGFP intensity of SeV-Pur2 cells transfected with the siNEG was set to 1.0, and relative fluorescence intensities of all cells are indicated. The means and SD from three replicate experiments are presented.
Fig 3
Fig 3. Effects of SeVdp-mediated C overexpression on SeVdp RNA synthesis.
(A) Inhibition of SeVdp mRNA synthesis by SeV C overexpression. The NP, L, and EGFP mRNA levels were determined by RT-qPCR. The mRNA levels of SeV-Pur cells were set to 1.0, and relative mRNA levels of all infected cells are indicated. As controls, the mRNA levels in non-infected cells (mock) were also determined. GAPDH expression was used to normalize the data. The means and SD (n = 3) are presented. (B) Inhibition of SeVdp genomic RNA synthesis by SeV C overexpression. The levels of genomic RNA were determined by RT-qPCR. The genomic RNA level of SeV-Pur cells was set to 1.0, and relative levels of the infected cells are indicated. Non-infected cells (mock) were used as a negative control. GAPDH expression was used to normalize the data. The means and SD (n = 3) are presented. (C) Reversal effect of P expression on C-mediated inhibition. SeV-C2 cells were transfected with an empty plasmid (control), either P or L expression plasmid (P or L), or both P and L expression plasmids (P + L). Two days after transfection, EGFP expression was measured by flow cytometry. The EGFP intensity of cells transfected with the empty plasmid was set to 1.0, and relative fluorescence intensities of all cells are indicated. The means and SD from three replicate experiments are presented. (D) Effects of siRNA against the L gene on elimination of SeVdp vectors from infected cells. SeV-Pur2 and SeV-C2 cells were transfected with siRNA against the L gene (siL527) or negative control siRNA (siNEG) on days 0, and 6, and NP-positive cells were determined by flow cytometry on day 13. NP-positive cells transfected with the siNEG were set to 100% (data not shown), and the percentages of NP-positive cells transfected with siL527 are indicated. The means and SD from three replicate experiments are presented.
Fig 4
Fig 4. Regulation of transgene expression by endogenous miRNAs.
(A) Genome structure of the SeVdp vector containing miRNA target sequences. Four copies of miRNA target sequence were incorporated into the 3′ UTR of both C-opt genes on the SeV-C2 backbone (SeV-C2-miRT). To construct SeV-C2-21T and SeV-C2-let7T, miR-21 and let-7a target sequences were incorporated into SeV-C2, respectively. (B) Effect of miR-21 on transgene expression mediated by SeVdp vectors. The EGFP expression levels of SeV-C2 cells and SeV-C2-21T cells were measured by flow cytometry. The EGFP intensity of SeV-C2 cells was set to 1.0, and relative intensity of SeV-C2-21T cells is indicated. The means and SD from three replicate experiments are presented. (C) Effect of antimiR-21 on transgene expression mediated by the SeVdp vector. SeV-C2-21T cells were transfected with 50 nM LNA scramble (AM-scr) or antimiR-21 (AM-21), and EGFP expression levels were measured by flow cytometry. The EGFP intensity of cells transfected with the AM-scr was set to 1.0, and relative intensity of cells transfected with AM-21 is indicated. The means and SD from three replicate experiments are presented. (D) Effect of let-7 on transgene expression mediated by SeVdp vectors. Normal human dermal fibroblasts (NHDF) or human iPS cells (hiPSC) were infected with SeV-Pur2, SeV-C2, or SeV-C2-let7T, and EGFP expression levels were measured by flow cytometry. The EGFP intensities of cells infected with SeV-Pur2 were set to 1.0, and relative fluorescence intensities of SeV-C2 and SeV-C2-let7T cells are indicated. The means and SD from three replicate experiments are presented.

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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.
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