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.
The past decade has seen the development and application of neural stem cells (NSCs) as a novel gene delivery vector for targeted cancer gene therapy from bench to bedside [
It has been described that miR-199a-5p, miR-199a-3p, and miR-214, which are coexpressed from the miR-199a/214 cluster on Chromosome 1, negatively regulate hypoxia-induced cell migration via downregulation of the HIF-1
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed as a powerful tool for targeted genome editing [
To expand the CRISPR toolkit for transcriptional regulation, a catalytically dead Cas9 (dCas9) is created by mutating both nuclease domains of Cas9 [
Here we have attempted to inhibit the miR-199a/214 cluster using a CRISPRi system to promote hiPS-NSC migration towards tumors under hypoxic condition. Our data showed that the CRISPRi system successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in hiPS-NSCs and significantly enhanced their tumor tropism in vitro and in vivo.
A nonintegrating human iPSC (epi-hiPSC) line was generated and characterized as previously described [
NSCs were derived from the epi-hiPSC line and characterized as previously described [
Mouse metastatic breast cancer cell line 4T1 was purchased from American Type Culture Collection (Manassas, VA, USA). 4T1-luc cell line was purchased from Caliper (Mountain View, CA, USA). 4T1 and 4T1-luc cell lines were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin, and 50
For AP staining, cells were fixed with 90% alcohol for 2 min and washed three times with PBS and stained with BCIP/NBT for 30 min in the darkness. For immunostaining, cells were fixed in 4.0% paraformaldehyde for 20 min, permeabilized with 0.5% Tween-20 for 30 min, incubated with primary antibody overnight, and incubated with secondary antibody (Invitrogen) for 1 hour. The nuclei were counterstained by DAPI. The cells were imaged with an inverted confocal microscope. The primary antibodies used in this study were OCT4 (1 : 500, Abcam, Cambridge, MA, USA), nestin (1 : 100, Millipore), GFAP (1 : 500, Millipore), and
The fragment containing the U6-Chimeric and CBh-hSpCas9 expression cassette from the pX330 plasmid was cloned into the pFastBac1 plasmid. To create the dCas9, the D10A and H840A mutations were introduced into the RuvC1 and HNH nuclease domains of the hSpCas9 gene using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The resulting plasmid was called pFB/dCas9 and used as a template to construct the baculovirus-delivered CRISPRi system.
To search an optimal sgRNA target site for CRISPRi, the sequence of the promoter region (−357 bp to −1 bp) containing an E-box element of the miR-199a/214 cluster was retrieved from the UCSC Genome Browser (
Baculoviral vectors carrying the CRISPRi system or the control construct were produced and propagated in Sf9 insect cells according to the Bac-to-Bac Baculovirus Expression system manual (Invitrogen). The hiPS-NSCs were transduced with the baculoviral vectors overnight at a multiplicity of infection of 100 plaque-forming units per cell in the NSC medium.
For microRNA qPCR, small RNA was isolated using PureLink microRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion, Austin, TX, USA). Then polyA tailing and cDNA synthesis were performed using Ncode VILO microRNA cDNA Synthesis Kit (Invitrogen). The forward primers for qPCR analysis were designed based on the known mature microRNA sequence, with additional 3 “A”s at the 3′ end to improve amplification specificity (Supplementary Table
In vitro migration assays used the 4T1 cells as an attractant and were performed in Boyden chambers with the BD Falcon HTS FluoroBlok 24-well Multiwell Insert System. 4T1 cells were seeded into a 24-well companion plates in Opti-MEM (Invitrogen) at a density of 2.5 × 105 cells per well. Only DMEM was used in the wells of the blank group. HiPS-NSCs were labeled with Calcein-AM (Invitrogen). The labeled hiPS-NSCs were suspended in Opti-MEM and seeded into the Boyden chamber transwell inserts at a density of 5 × 104 cells per insert. After 24 h of culture at 37°C under normoxic or hypoxic conditions, the fluorescent hiPS-NSCs on the bottom side of the inserts (represent migrating cells) were counted and the migration rate was calculated. All experiments were conducted in 6 replicates.
All animal work was done in accordance with a protocol approved by the Institutional Animal Care and Use Committee. To access the in vivo tumor tropism of the hiPS-NSCs, a mouse model of breast cancer lung metastasis was established as previously described [
All data are represented as mean ± SD. The statistical significance of differences was determined by paired, two-tailed Student’s
An epi-hiPSC line was created from foreskin fibroblasts by electroporation of episomal vectors expressing reprogramming factors Oct4, Sox2, Klf4, L-Myc, and Lin28. The epi-hiPSC line was characterized by AP staining, immunostaining for the pluripotency marker Oct4, and embryoid body differentiation (Figure
Deviation of NSCs from an epi-hiPSC line. (a) Characterization of an epi-hiPSC line generated with nonintegrating episomal vectors. From left to right: colony morphology, AP staining, immunostaining of Oct4, and embryoid body differentiation. (b) Characterization and in vitro neural differentiation of hiPS-NSCs. From left to right: bipolar cell morphology, immunostaining of nestin (NSC early stage marker), GFAP (astrocyte marker), and
In the view that miR-199a-5p, miR-199a-3p, and miR-214 of the miR-199a/214 cluster are targeting the HIF-1
Targeted inhibition of miR-199a/214 cluster using a CRISPRi system in NSCs. (a) Diagram showing miR-199a-5p, miR-199a-3p, and miR-214 of the miR-199a/214 cluster are targeting multiple important components in the signaling pathways that mediate hypoxia-induced migration of NSCs. (b) Schematic representation showing blockage of transcription of the miR-199a/214 cluster using a dCas9 targeting the E-box in the promoter region. The sequence of the gRNA target site is shown and the PAM sequence is underlined. The E-box sequence is highlighted in red. (c) Absolute expression levels of miR-199a-5p, miR-199a-3p, and miR-214 in NSCs are quantified by qPCR (
Baculovirus was employed to efficiently deliver the CRISPRi system targeting the E-box element or the control vector into hiPS-NSCs. MicroRNA qPCR was performed to access the absolute expression levels of miR-199a-5p, miR-199a-3p, and miR-214 in hiPS-NSCs transduced with the CRISPRi system and under hypoxic condition (Figure
We then further investigated whether the inhibition of miR-199a/214 cluster could derepress the hypoxia-related signaling pathways and promote hiPS-NSC migration under hypoxic condition. The mRNA expression levels of the reported microRNA targets, HIF1A, MET, and MAPK1, were measured by qPCR. The CXCR4 was included as it was another important target of HIF-1
Inhibition of the miR-199a/214 cluster derepresses hypoxia-related targets and promotes NSC tumor tropism in vitro. (a) Relative expression levels of HIF1A, MET, MAPK1, and CXCR4 in NSCs are quantified by qPCR (
Next, the in vitro migratory capacity of hiPS-NSCs towards 4T1, a mouse metastatic breast cancer cell line, was evaluated by Boyden chamber cell migration assays (Figure
To further examine whether inhibition of the miR-199a/214 cluster could improve the hiPS-NSC tumor tropism in vivo, an animal study was performed using a mouse model of breast cancer lung metastasis. In brief, 4T1-luc metastatic breast cancer cells were inoculated into the BALB/c mice via tail vein injection. After 1 week, lung metastases were established and the mice were divided into 2 groups randomly. Then hiPS-NSCs transduced with the CRISPRi system or the control vector were labeled by DiR and injected into the mice through the tail vein, respectively. The next day, whole-body luminescent and fluorescent images were taken to confirm the presence of 4T1-luc tumors and DiR-labeled hiPS-NSCs (Figure
Inhibition of the miR-199a/214 cluster enhances NSC tumor tropism in vivo. (a) Diagram showing the protocol of NSC in vivo tumor tropism assay. (b) Representative ex vivo organs images showing the tumor tropism of NSCs in mice at day 8. The luminescent images (upper panels) show luc-4T1 tumor metastases in the lung. The fluorescent images (lower panels) show the organ distribution of NSCs. The organs shown in each panel from left to right: lung, liver, spleen, kidney, heart, brain, stomach, spinal cord, and femur. (c) Histogram showing the percentages of NSCs distributed in the lungs, the livers, and the spleens (
The poor prognosis of malignant tumors is largely due to the ability of these tumors to infiltrate in the human body and the ineffectiveness of conventional therapies, such as surgery and radio- and chemotherapy, to eliminate disseminated tumors. Apparently, there is an urgent need to develop novel therapeutic approaches to overcome the limitations of current cancer therapies. NSCs are able to home in on not only the brain tumors but also solid tumors of a nonneural origin [
The great potential of NSCs in cancer gene therapy underscores the importance of a robust, reliable source for the large scale, standardized production of human NSCs that meet the requirements of good clinical practice. The availability of hiPSC technique has provided an accessible, stable source to produce unlimited amounts of NSCs for cell therapies [
To our knowledge, this is the first attempt to use baculovirus to deliver a CRISPRi system into NSCs. The baculovirus is a kind of insect virus but able to transduce human cells efficiently. Unlike traditionally used human viral vectors, such as retrovirus, adenovirus, and adeno-associated virus, baculovirus is inherently incapable of replicating in human cells and there is no detectable preexisting immunity to baculovirus in human; hence, it has been extensively exploited as a novel gene delivery vector for clinical applications [
More importantly, the animal experiment using a breast cancer lung metastasis mouse model has demonstrated that targeted inhibition of the miR-199a/214 cluster by CRISPRi significantly augmented the tumor tropism of hiPS-NSCs in vivo. The tumor microenvironment is usually hypoxic due to the high proliferation rate of tumor cells. The tumor cells will release cytokines such SDF-1 and HGF to stimulate angiogenesis to support tumor growth. After intravenous injection, the NSC vectors will travel through the organs via the circulation system. They are supposed to sense the SDF-1 and HGF gradient and migrate through the venular walls into the tumor tissues. However, the SDF-1/CXCR4 and HGF/c-Met signaling pathways are negatively regulated by the miR-199a/214 cluster in NSCs. In our animal experiment, we demonstrated that, by inhibition of the miR-199a/214 cluster, a significantly higher ratio of NSC vectors migrated into the tumor tissues. In previous animal study, it has been observed that off-target therapeutic gene expression by NSC vectors would cause serious damage to healthy organs [
We have developed a baculovirus-delivered CRISPRi system that specifically inhibits the miR-199a/214 cluster on Chromosome 1 by blocking the E-box in the promoter region. Targeted inhibition of the miR-199a/214 cluster in NSCs significantly improved their tumor tropism in vitro and in vivo.
All authors have no conflict of interests to declare.
Yumei Luo and Xuehu Xu contributed equally to this work.
This research was supported by the National Natural Science Foundation of China (81401206, 31171229, and U1132005), Science and Technology Planning Projects of Guangdong Province, China (2016A030308011, 2016B090918130, 2014A020212357, 2014A030312012, and 2013B051000087), Science and Information Technology Key Project of Guangzhou City (201508020258, 201400000003-4, and 201400000004-4), and Guangzhou City Medical Science and Technology Program (20141A011091).