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Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

Roos AK, Eriksson F, Timmons JA, Gerhardt J, Nyman U, Gudmundsdotter L, Bråve A, Wahren B, Pisa P - PLoS ONE (2009)

Bottom Line: Electrical pulses have been used to enhance uptake of molecules into living cells for decades.In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression.This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Pathology, Cancer Center Karolinska R8:01, Immune and Gene Therapy Laboratory, Karolinska Institute, Stockholm, Sweden. roos.anki@gmail.com

ABSTRACT

Background: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood.

Methodology/principal findings: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid.

Conclusions/significance: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

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Related in: MedlinePlus

β-galactosidase expression in skin following DNA electrovaccination.Mice were injected intradermally with 20 ug DNA encoding LacZ and the injection site was electroporated. (a, b) Skin biopsies were removed after 24 hrs and stained with X-gal (a); arrows indicate needle penetration sites (b). (c–g) Distribution of β-galactosidase expressing cells in skin sections 24 hrs after DNA electrovaccination. Skin sections were prepared for histochemistry and stained with X-gal; 4X (c, e, g) and 20X (d, f) magnifications of skin sections show β-galactosidase expressing cells around the panniculus carnosus muscle layer (c–d) and in the hypodermis (c–e), dermis and epidermis (e, f). Untreated skin stained with X-gal was used as a negative control (g). P = panniculus carnosus, H = hypodermis, D = dermis and E = epidermis.
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pone-0007226-g002: β-galactosidase expression in skin following DNA electrovaccination.Mice were injected intradermally with 20 ug DNA encoding LacZ and the injection site was electroporated. (a, b) Skin biopsies were removed after 24 hrs and stained with X-gal (a); arrows indicate needle penetration sites (b). (c–g) Distribution of β-galactosidase expressing cells in skin sections 24 hrs after DNA electrovaccination. Skin sections were prepared for histochemistry and stained with X-gal; 4X (c, e, g) and 20X (d, f) magnifications of skin sections show β-galactosidase expressing cells around the panniculus carnosus muscle layer (c–d) and in the hypodermis (c–e), dermis and epidermis (e, f). Untreated skin stained with X-gal was used as a negative control (g). P = panniculus carnosus, H = hypodermis, D = dermis and E = epidermis.

Mentions: As in vivo monitoring of luciferase expression does not show individual cells and the light emission spreads beyond the location of transfected cells, histochemical staining of β-galactosidase was used to investigate the location of the DNA transfected cells in the skin. This demonstrated that the use of needle array electrodes produced a uniform transfection of the treated volume, resulting in a large area of transfected cells confined to the space between the needle rows (Fig. 2a), with the majority of the transfected cells concentrated around the panniculus carnosus muscle layer in the deepest layer of the skin (Fig. 2c–e). Transfected cells were also found in the hypodermis (Fig. 2c–e) and in sparse numbers through the dermis and epidermis (Fig. 2e, f). However, no expression was observed in actual muscle cells. No background staining was seen in untreated skin (Fig. 2g) and neither luciferase nor LacZ expression was detected in the tissue immediately below the injected skin (data not shown). The data demonstrate that intradermal DNA delivery in combination with electroporation results in cell transfection throughout all layers of the skin and yields faster, higher and more consistent gene expression, compared to intradermal gene delivery alone.


Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

Roos AK, Eriksson F, Timmons JA, Gerhardt J, Nyman U, Gudmundsdotter L, Bråve A, Wahren B, Pisa P - PLoS ONE (2009)

β-galactosidase expression in skin following DNA electrovaccination.Mice were injected intradermally with 20 ug DNA encoding LacZ and the injection site was electroporated. (a, b) Skin biopsies were removed after 24 hrs and stained with X-gal (a); arrows indicate needle penetration sites (b). (c–g) Distribution of β-galactosidase expressing cells in skin sections 24 hrs after DNA electrovaccination. Skin sections were prepared for histochemistry and stained with X-gal; 4X (c, e, g) and 20X (d, f) magnifications of skin sections show β-galactosidase expressing cells around the panniculus carnosus muscle layer (c–d) and in the hypodermis (c–e), dermis and epidermis (e, f). Untreated skin stained with X-gal was used as a negative control (g). P = panniculus carnosus, H = hypodermis, D = dermis and E = epidermis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748717&req=5

pone-0007226-g002: β-galactosidase expression in skin following DNA electrovaccination.Mice were injected intradermally with 20 ug DNA encoding LacZ and the injection site was electroporated. (a, b) Skin biopsies were removed after 24 hrs and stained with X-gal (a); arrows indicate needle penetration sites (b). (c–g) Distribution of β-galactosidase expressing cells in skin sections 24 hrs after DNA electrovaccination. Skin sections were prepared for histochemistry and stained with X-gal; 4X (c, e, g) and 20X (d, f) magnifications of skin sections show β-galactosidase expressing cells around the panniculus carnosus muscle layer (c–d) and in the hypodermis (c–e), dermis and epidermis (e, f). Untreated skin stained with X-gal was used as a negative control (g). P = panniculus carnosus, H = hypodermis, D = dermis and E = epidermis.
Mentions: As in vivo monitoring of luciferase expression does not show individual cells and the light emission spreads beyond the location of transfected cells, histochemical staining of β-galactosidase was used to investigate the location of the DNA transfected cells in the skin. This demonstrated that the use of needle array electrodes produced a uniform transfection of the treated volume, resulting in a large area of transfected cells confined to the space between the needle rows (Fig. 2a), with the majority of the transfected cells concentrated around the panniculus carnosus muscle layer in the deepest layer of the skin (Fig. 2c–e). Transfected cells were also found in the hypodermis (Fig. 2c–e) and in sparse numbers through the dermis and epidermis (Fig. 2e, f). However, no expression was observed in actual muscle cells. No background staining was seen in untreated skin (Fig. 2g) and neither luciferase nor LacZ expression was detected in the tissue immediately below the injected skin (data not shown). The data demonstrate that intradermal DNA delivery in combination with electroporation results in cell transfection throughout all layers of the skin and yields faster, higher and more consistent gene expression, compared to intradermal gene delivery alone.

Bottom Line: Electrical pulses have been used to enhance uptake of molecules into living cells for decades.In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression.This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Pathology, Cancer Center Karolinska R8:01, Immune and Gene Therapy Laboratory, Karolinska Institute, Stockholm, Sweden. roos.anki@gmail.com

ABSTRACT

Background: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood.

Methodology/principal findings: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid.

Conclusions/significance: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

Show MeSH
Related in: MedlinePlus