title: Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition.
abstract: The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.
authors.affiliation: Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China; Laboratory of Membrane Biology, New York Blood Center, New York, NY 10065, USA.
authors.forename: Yumei
authors.identifier: 0000-0002-4175-1906
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Gastrointestinal Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China.
authors.forename: Xuehu
authors.initials: X
authors.lastname: Xu
authors.affiliation: Laboratory of Membrane Biology, New York Blood Center, New York, NY 10065, USA.
authors.forename: Xiuli
authors.initials: X
authors.lastname: An
authors.affiliation: Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education Institutes, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China.
authors.forename: Xiaofang
authors.identifier: 0000-0002-5758-046X
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore 117543.
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065, USA.
authors.forename: Detu
authors.identifier: 0000-0002-9268-1012
authors.initials: D
authors.lastname: Zhu