Combinatorial control of transgene expression by hypoxia-responsive promoter and microrna regulation for neural stem cell-based cancer therapy - PubMed (original) (raw)

Combinatorial control of transgene expression by hypoxia-responsive promoter and microrna regulation for neural stem cell-based cancer therapy

Yumei Luo et al. Biomed Res Int. 2014.

Abstract

Owing to their strong migratory capacity, tumor tropism, and tumor inhibitory effect, neural stem cells (NSCs) have recently emerged as one of the most attractive gene delivery vectors for cancer therapy. However, further animal studies found that proportional NSC vectors were distributed to nontarget organs after intravenous injection and the nonspecific transgene expression led to significant cytotoxic effects in these organs. Hence, an expression cassette that controls the transgene expression within NSC vectors in a tumor site-specific manner is desired. Considering hypoxia as a hallmark of tumor microenvironment, we have developed a novel NSC vector platform coupling transcriptional targeting with microRNA (miRNA) regulation for tumor hypoxia targeting. This combinatorial vector employed a hypoxia-responsive promoter and repeated targeting sequences of an miRNA that is enriched in NSCs but downregulated upon hypoxia induction to control the transgene expression. This resulted in significantly improved hypoxic selectivity over the use of a control vector without miRNA regulation. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of NSC vectors with high targeting specifcity for cancer therapy.

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Figures

Figure 1

iPS cell line characterization and neural differentiation in vitro. (a) iPS cell line characterization. From left to right: iPS cell colony, AP staining, immunostaining to show protein expression of embryonic stem cell markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. (b) Neural differentiation in vitro. From left to right: immunostaining to show protein expression of NSC early stage marker nestin, NSC differentiation markers GFAP (glial cell marker), and _β_-III tubulin (neuron marker).

Figure 2

NSCs response to tumor hypoxia. (a) mRNA expression level of hypoxia-related signal receptors, c-Met, CXCR4, c-Kit, and VEGFR2, in NSCs cultured 24 h under normoxic and hypoxic conditions is quantified by qPCR. (b) Migration rates of NSCs towards MCF-7 breast cancer cells under normoxic and hypoxic conditions are quantified by Boyden chamber cell migration assays. MCF-7 cells were seeded in the lower chamber, and blank medium in the lower chamber was used for the negative control group. NSCs were stained with calcein-AM and seeded in the upper chamber. After 24 h, the labelled NSCs which migrated toward the lower chamber were evaluated (n = 5). Top: percentage of migrated NSCs. Bottom: fluorescence images showing the migration of NSCs toward the lower chambers. Error bars: s.d. *P < 0.05, **P < 0.01.

Figure 3

Selection of the optimal hypoxia-responsive promoter and normoxia-specific miRNA in NSCs. (a) Luciferase assays showing promoter activities. CXCR4 promoter, VEGF promoter, optHRP, and EF1_α_ promoter were cloned into the pGL4.11 promoterless luciferase reporter plasmids, respectively. The promoterless pGL4.11 plasmid was included as negative control. NSCs were divided into 5 groups, transfected with the above constructs, and cultured 24 h under normoxic and hypoxic conditions, respectively. Then the promoter activities were quantified by luciferase assays. (b) Absolute expression levels of miR-199a-5p in NSCs under normoxic and hypoxic conditions are quantified by qPCR. miRNA copy numbers were calculated based on a standard curve generated using a synthetic LIN-4 RNA oligonucleotide. Abbreviation: RLU, relative luminescence unit. Error bars: s.d. *P < 0.05, **P < 0.01.

Figure 4

Combinatorial effect of optHRP and miR-199a-5p on transgene regulation in NSC. (a) Schematic representation of the combinatorial expression cassettes containing the optHRP promoter and miRNA target sequences. optHRP, an artificially optimized hypoxia-responsive promoter; luc, luciferase reporter gene; miRNA target sequences as detailed in Table 1 were inserted into the 3′UTR; pA, polyA signal. (b) Transgene expression levels of different expression cassettes within NSCs under normoxic and hypoxic conditions are quantified by luciferase assays. Abbreviation: RLU, relative luminescence unit. Error bars: s.d. *P < 0.05, **P < 0.01.

Figure 5

In vivo transgene expression in MCF-7 tumor-bearing mice. (a) Schematic representation of the in vivo expression assay. MCF-7 cells are inoculated into the right mammary fat pad of the mouse and sham injections are given on the contralateral side. After the tumors develop, NSC vectors are inoculated into the tumor sites and sham injection sites, respectively. 24 h after NSC inoculation, luciferase reporter gene expression levels are monitored by live animal imaging. (b) Bioluminescent image showing luciferase reporter gene expression in the tumor-bearing mice. Red circles indicate the inoculation sites of NSC vectors. (c) Average quantitative transgene expression levels in the tumor sites and sham injection sites. Error bars: s.d. *P < 0.05, **P < 0.01.

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Combinatorial control of transgene expression by hypoxia-responsive promoter and microrna regulation for neural stem cell-based cancer therapy - PubMed (original) (raw)