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


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.

SUBMITTER: Hsu MN 

PROVIDER: S-EPMC6735509 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Hsu Mu-Nung MN   Liao Han-Tsung HT   Truong Vu Anh VA   Huang Kai-Lun KL   Yu Fu-Jen FJ   Chen Hwei-Hsien HH   Nguyen Thi Kieu Nuong TKN   Makarevich Pavel P   Parfyonova Yelena Y   Hu Yu-Chen YC  

Theranostics 20190814 21


<b>Background</b>: 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. <b>Methods</b>: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (<i>  ...[more]

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