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Development and characterization of an enhanced nonviral expression vector for electroporation cancer treatment.

Forde PF, Hall LJ, Sadadcharam M, de Kruijf M, O' Sullivan GC, Soden DM - Mol Ther Methods Clin Dev (2014)

Bottom Line: However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches.We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types.Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model.

View Article: PubMed Central - PubMed

Affiliation: Cork Cancer Research Centre, Leslie C Quick Laboratory, BioSciences Institute, University College Cork , Cork, Ireland.

ABSTRACT
Nonviral plasmid DNA gene therapy represents a promising approach for the treatment of many diseases including cancer. Intracellular delivery of DNA can be achieved with the application of electroporation, which facilitates the initial transport of exogenous DNA across the cell membrane into the cytoplasm. However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches. To overcome these expression difficulties, we developed a proof-of-concept vector enhanced expression vector (EEV), which incorporates elements from viral systems including nuclear localization sequences and a viral replicase from the Semliki Forest virus. The replicase allows for cytoplasmic mRNA expression and bypasses the need for nuclear localization to generate high levels of gene expression. We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types. Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model. Using a novel endoscopic electroporation system, EndoVe, we demonstrate and compare, for the first time, both standard cytomegalovirus (CMV) promoter-driven plasmid and EEV gene expression in intraluminal porcine tissues. Our EEV plasmid displays reliable and superior expression capability, and due to its inherent induced oncolytic activity in transfected cells, it may enhance the efficacy and safety of several cancer immunogene therapy approaches.

No MeSH data available.


Related in: MedlinePlus

β-galactosidase expression in murine tissue. (a) Copy numbers of lacZ transgene ascertained by quantitative polymerase chain reaction (PCR) in murine tissue. Bar graph showing absolute copy number of the lacz transgene per nanogram of genomic DNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All gDNA samples were normalized to 100 ng of DNA prior to PCR. Each individual sample was analyzed in triplicate for each quantiative PCR (qPCR). qPCR values are means ± standard error of the mean (SEM) of triplicate measurements. A comparison of enhanced expression vector (pEEV) (light gray bars) and pCMV (dark gray bars) samples were performed. ***P < 0.0001. (b) Evaluation of lacZ mRNA transcript expression by qRT-PCR in murine tissue. Bar graph presenting the relative expression of the lacZ mRNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All qPCR data were normalized using 18S RNA as reference gene. Relative expression levels are plotted as means ± SEM of triplicate measurements. pEEV (light gray bars) and pCMV (dark gray bars). pEEV lacZ expressed was significantly higher than pCMV lacZ. ***P < 0.0001.
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fig2: β-galactosidase expression in murine tissue. (a) Copy numbers of lacZ transgene ascertained by quantitative polymerase chain reaction (PCR) in murine tissue. Bar graph showing absolute copy number of the lacz transgene per nanogram of genomic DNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All gDNA samples were normalized to 100 ng of DNA prior to PCR. Each individual sample was analyzed in triplicate for each quantiative PCR (qPCR). qPCR values are means ± standard error of the mean (SEM) of triplicate measurements. A comparison of enhanced expression vector (pEEV) (light gray bars) and pCMV (dark gray bars) samples were performed. ***P < 0.0001. (b) Evaluation of lacZ mRNA transcript expression by qRT-PCR in murine tissue. Bar graph presenting the relative expression of the lacZ mRNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All qPCR data were normalized using 18S RNA as reference gene. Relative expression levels are plotted as means ± SEM of triplicate measurements. pEEV (light gray bars) and pCMV (dark gray bars). pEEV lacZ expressed was significantly higher than pCMV lacZ. ***P < 0.0001.

Mentions: The EEV plasmid (15.462 kb) is approximately three times larger, than the standard pCMV plasmid (6.233 kb), which could impact on transfection efficacy and expression. To establish this, we compared the transfection efficiency in vivo of pEEV with the pCMV plasmid. In order to determine efficiency, copy numbers of each plasmid expressing the lacZ transgene delivered by electroporation were measured. Both plasmids were detected in all the tissues tested. In general, they were significantly more (P < 0.001) copies of pCMV than pEEV (Figure 2a), with the spleen and tumor having highest plasmid transfection.


Development and characterization of an enhanced nonviral expression vector for electroporation cancer treatment.

Forde PF, Hall LJ, Sadadcharam M, de Kruijf M, O' Sullivan GC, Soden DM - Mol Ther Methods Clin Dev (2014)

β-galactosidase expression in murine tissue. (a) Copy numbers of lacZ transgene ascertained by quantitative polymerase chain reaction (PCR) in murine tissue. Bar graph showing absolute copy number of the lacz transgene per nanogram of genomic DNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All gDNA samples were normalized to 100 ng of DNA prior to PCR. Each individual sample was analyzed in triplicate for each quantiative PCR (qPCR). qPCR values are means ± standard error of the mean (SEM) of triplicate measurements. A comparison of enhanced expression vector (pEEV) (light gray bars) and pCMV (dark gray bars) samples were performed. ***P < 0.0001. (b) Evaluation of lacZ mRNA transcript expression by qRT-PCR in murine tissue. Bar graph presenting the relative expression of the lacZ mRNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All qPCR data were normalized using 18S RNA as reference gene. Relative expression levels are plotted as means ± SEM of triplicate measurements. pEEV (light gray bars) and pCMV (dark gray bars). pEEV lacZ expressed was significantly higher than pCMV lacZ. ***P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362347&req=5

fig2: β-galactosidase expression in murine tissue. (a) Copy numbers of lacZ transgene ascertained by quantitative polymerase chain reaction (PCR) in murine tissue. Bar graph showing absolute copy number of the lacz transgene per nanogram of genomic DNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All gDNA samples were normalized to 100 ng of DNA prior to PCR. Each individual sample was analyzed in triplicate for each quantiative PCR (qPCR). qPCR values are means ± standard error of the mean (SEM) of triplicate measurements. A comparison of enhanced expression vector (pEEV) (light gray bars) and pCMV (dark gray bars) samples were performed. ***P < 0.0001. (b) Evaluation of lacZ mRNA transcript expression by qRT-PCR in murine tissue. Bar graph presenting the relative expression of the lacZ mRNA 2 days after electroporation obtained from MF1-nu/nu mouse tissue. All qPCR data were normalized using 18S RNA as reference gene. Relative expression levels are plotted as means ± SEM of triplicate measurements. pEEV (light gray bars) and pCMV (dark gray bars). pEEV lacZ expressed was significantly higher than pCMV lacZ. ***P < 0.0001.
Mentions: The EEV plasmid (15.462 kb) is approximately three times larger, than the standard pCMV plasmid (6.233 kb), which could impact on transfection efficacy and expression. To establish this, we compared the transfection efficiency in vivo of pEEV with the pCMV plasmid. In order to determine efficiency, copy numbers of each plasmid expressing the lacZ transgene delivered by electroporation were measured. Both plasmids were detected in all the tissues tested. In general, they were significantly more (P < 0.001) copies of pCMV than pEEV (Figure 2a), with the spleen and tumor having highest plasmid transfection.

Bottom Line: However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches.We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types.Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model.

View Article: PubMed Central - PubMed

Affiliation: Cork Cancer Research Centre, Leslie C Quick Laboratory, BioSciences Institute, University College Cork , Cork, Ireland.

ABSTRACT
Nonviral plasmid DNA gene therapy represents a promising approach for the treatment of many diseases including cancer. Intracellular delivery of DNA can be achieved with the application of electroporation, which facilitates the initial transport of exogenous DNA across the cell membrane into the cytoplasm. However, it does not guarantee further transport of the DNA from the cytoplasm to the nucleus for subsequent mRNA expression, resulting in varying degrees of exogenous gene translation and a major limitation in comparison to viral approaches. To overcome these expression difficulties, we developed a proof-of-concept vector enhanced expression vector (EEV), which incorporates elements from viral systems including nuclear localization sequences and a viral replicase from the Semliki Forest virus. The replicase allows for cytoplasmic mRNA expression and bypasses the need for nuclear localization to generate high levels of gene expression. We have demonstrated that our EEV is capable of achieving high levels of expression in a variety of tissue types. Antitumor effects of pEEV were demonstrated by the delayed growth and increased survival of the nontherapeutic pEEV-treated CT26 tumor model. Using a novel endoscopic electroporation system, EndoVe, we demonstrate and compare, for the first time, both standard cytomegalovirus (CMV) promoter-driven plasmid and EEV gene expression in intraluminal porcine tissues. Our EEV plasmid displays reliable and superior expression capability, and due to its inherent induced oncolytic activity in transfected cells, it may enhance the efficacy and safety of several cancer immunogene therapy approaches.

No MeSH data available.


Related in: MedlinePlus