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Theranostics 2019; 9(21):6099-6111. doi:10.7150/thno.36790 This issue Cite

Research Paper

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration

Mu-Nung Hsu^1,*, Han-Tsung Liao^2,3,4,*, Vu Anh Truong¹, Kai-Lun Huang¹, Fu-Jen Yu¹, Hwei-Hsien Chen⁵, Thi Kieu Nuong Nguyen¹, Pavel Makarevich⁶, Yelena Parfyonova^7,8, Yu-Chen Hu^1,9,✉

1. Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, 300
2. Department of Plastic and Reconstructive Surgery, Craniofacial Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan, 333
3. College of Medicine, Chang Gung University, Taoyuan, Taiwan, 333
4. Department of Plastic surgery, Xiamen Chang Gung hospital, China 361028
5. Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, 350
6. Laboratory of Gene and Cell Therapy, Institute for Regenerative Medicine, Lomonosov Moscow State University, Moscow, Russia 119192
7. Laboratory of Angiogenesis, National Medical Research Center for Cardiology, Moscow, Russia 121152
8. Laboratory of Postgenomic Technologies in Medicine, Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia 119192
9. Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan 300
* These two authors contributed equally to this work

✉ Corresponding author: Tel: +886-3-5718245; Fax: +886-3-5715408; E-mail address: ychunthu.edu.tw

Abstract

Background: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration.

Methods: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (BDNF, GDNF and NGF). The hybrid BV was used to transduce rat adipose-derived stem cells (ASC) and functionalize the ASC sheets. We further implanted the ASC sheets into sciatic nerve injury sites in rats.

Results: Transduction of rat ASC with the hybrid BV vector enabled robust, simultaneous and prolonged activation of the 3 neurotrophic factors for at least 21 days. The CRISPRa-engineered ASC sheets were able to promote Schwann cell (SC) migration, neuron proliferation and neurite outgrowth in vitro. The CRISPRa-engineered ASC sheets further enhanced in vivo functional recovery, nerve reinnervation, axon regeneration and remyelination.

Conclusion: These data collectively implicated the potentials of the hybrid BV-delivered CRISPRa system for multiplexed activation of endogenous neurotrophic factor genes in ASC sheets to promote peripheral nerve regeneration.

Keywords: CRISPR activation, adipose-derived stem cell, baculovirus, cell sheet, neurotrophic factor, nerve regeneration

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