RNA Virus-Based Episomal Vector with a Fail-Safe Switch Facilitating Efficient Genetic Modification and Differentiation of iPSCs

Yumiko Komatsu,Dan Takeuchi,Tomoya Tokunaga,Hidetoshi Sakurai,Akiko Makino,Tomoyuki Honda,Yasuhiro Ikeda,Keizo Tomonaga

doi:10.1016/j.omtm.2019.05.010

Abstract

A gene delivery system that allows efficient and safe stem cell modification is critical for next-generation stem cell therapies. An RNA virus-based episomal vector (REVec) is a gene transfer system developed based on Borna disease virus (BoDV), which facilitates persistent intranuclear RNA transgene delivery without integrating into the host genome. In this study, we analyzed susceptibility of human induced pluripotent stem cell (iPSC) lines from different somatic cell sources to REVec, along with commonly used viral vectors, and demonstrated highly efficient REVec transduction of iPSCs. Using REVec encoding myogenic transcription factor MyoD1, we further demonstrated potential application of the REVec system for inducing differentiation of iPSCs into skeletal muscle cells. Of note, treatment with a small molecule, T-705, completely eliminated REVec in persistently transduced cells. Thus, the REVec system offers a versatile toolbox for stable, integration-free iPSC modification and trans-differentiation, with a unique switch-off mechanism.

Highlights

Gene transfer technology that allows efficient and stable genetic modification of induced pluripotent stem cells is a key component in developing a successful gene therapy
To further investigate the efficiency of gene transfer into induced pluripotent stem cell (iPSC) derived from different somatic cell sources, seven human iPSC lines were plated on vitronectin coat as single cells and transduced with replication-defective RNA virus-based episomal vector (REVec) lacking the translation initiation codons for the glycoprotein gene[10] at a MOI of 1.0
To further quantify these results, qRT-PCR analysis was conducted and showed a decrease in expression of the pluripotency marker (OCT4) and an increase in expression of the mesoderm marker (BMP2), ectoderm marker (SOX1), and endoderm marker (SOX17) in embryoid body (EB), compared to that in iPSCs (Figure 3C). These results indicate that non-integrating REVec achieves efficient gene transfer into iPSCs and that transgene expression is maintained upon differentiation

Summary

Introduction

Gene transfer technology that allows efficient and stable genetic modification of induced pluripotent stem cells (iPSCs) is a key component in developing a successful gene therapy. The use of integrating viral vectors offer stable transgene expression in iPSCs, the risks of insertional mutagenesis and oncogenesis are recognized as major safety concerns surrounding these vectors.[1] Non-integrating viral vectors provide transient transgene expression in proliferating cells and, may not be suitable for long-term expression in iPSCs.[2,3] the efficiency of gene editing and off-target modifications represent major challenges in translating non-viral gene editing technologies for therapeutic applications.[4] Another important feature for a safe gene delivery system is an ability to regulate transgene expression, or a suicide switch, as a fail-safe mechanism. Development of an episomal vector system with an integrated safety switch would pave a new path for safe gene therapy applications

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Conclusion