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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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Related in: MedlinePlus

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.
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fig4: 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.

Mentions: To employ both hypoxia-responsive promoter and miRNA regulation to restrict transgene expression to hypoxic condition, we constructed a combinatorial expression cassette (Figure 4(a)). We used the optHRP-luc plasmid generated in the previous promoter activity experiment and introduced perfectly complementary miRNA target sequences of miR-199a-5p into the 3′UTR of the luciferase reporter gene (optHRP-luc-mir199a5pT). To rule out the possibility of less favorable transcription caused by introduction of a long repeat sequence into the 3′UTR, we constructed a control plasmid optHRP-luc-ScrT by replacing the miR-199a-5p target sequences with a scramble target sequence of the same length, which was designed based on the lack of significant similarity to any known miRNA (Table 1).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016878&req=5

fig4: 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.
Mentions: To employ both hypoxia-responsive promoter and miRNA regulation to restrict transgene expression to hypoxic condition, we constructed a combinatorial expression cassette (Figure 4(a)). We used the optHRP-luc plasmid generated in the previous promoter activity experiment and introduced perfectly complementary miRNA target sequences of miR-199a-5p into the 3′UTR of the luciferase reporter gene (optHRP-luc-mir199a5pT). To rule out the possibility of less favorable transcription caused by introduction of a long repeat sequence into the 3′UTR, we constructed a control plasmid optHRP-luc-ScrT by replacing the miR-199a-5p target sequences with a scramble target sequence of the same length, which was designed based on the lack of significant similarity to any known miRNA (Table 1).

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