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

Luo Y, Zhu D - Biomed Res Int (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, 63 Duobao Road, Liwan District, Guangzhou City, Guangdong Province 510150, China.

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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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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fig5: 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.

Mentions: We next investigated whether the combinatorial NSC vectors can mediate tumor site-specific transgene expression in vivo. An orthotopic mouse model of breast cancer was established by inoculating MCF-7 breast cancer cells into the right mammary fat pad and sham into the left (Figure 5(a)). After the tumor developed, NSCs transfected with the optHRP-luc-mir199a5pT plasmid were injected into the tumor sites and sham sites, respectively. The next day, luciferase reporter gene expression levels were monitored by a live animal bioimaging system (Figure 5(b)). Quantitative results from all the three mice were summarized in Figure 5(c), showing that NSC vectors displayed induced luciferase gene expressions in tumor sites by averagely 30 folds higher than those in sham sites. Thus, the combinatorial NSC vectors are demonstrated to be capable of mediating tumor site-specific transgene expression in vivo.


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

Luo Y, Zhu D - Biomed Res Int (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016878&req=5

fig5: 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.
Mentions: We next investigated whether the combinatorial NSC vectors can mediate tumor site-specific transgene expression in vivo. An orthotopic mouse model of breast cancer was established by inoculating MCF-7 breast cancer cells into the right mammary fat pad and sham into the left (Figure 5(a)). After the tumor developed, NSCs transfected with the optHRP-luc-mir199a5pT plasmid were injected into the tumor sites and sham sites, respectively. The next day, luciferase reporter gene expression levels were monitored by a live animal bioimaging system (Figure 5(b)). Quantitative results from all the three mice were summarized in Figure 5(c), showing that NSC vectors displayed induced luciferase gene expressions in tumor sites by averagely 30 folds higher than those in sham sites. Thus, the combinatorial NSC vectors are demonstrated to be capable of mediating tumor site-specific transgene expression in vivo.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University, 63 Duobao Road, Liwan District, Guangzhou City, Guangdong Province 510150, China.

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.

Show MeSH
Related in: MedlinePlus