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Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Bottom Line: Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.


Histological analysis reveals rapid GFP expression. Histological analysis of GFP expression in a time course (1-hour post treatment to day 21) after ID plasmid administration followed by EP with SEP in guinea pig skin. Skin biopsies were removed, cryosectioned, DAPI stained and visualized under fluorescence microscopy (10×). An injection only control is also shown. Photos are representative examples of multiple treatments. A region of the 2-hour time point section image is enlarged to allow the annotation of skin structures.
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vaccines-01-00384-f002: Histological analysis reveals rapid GFP expression. Histological analysis of GFP expression in a time course (1-hour post treatment to day 21) after ID plasmid administration followed by EP with SEP in guinea pig skin. Skin biopsies were removed, cryosectioned, DAPI stained and visualized under fluorescence microscopy (10×). An injection only control is also shown. Photos are representative examples of multiple treatments. A region of the 2-hour time point section image is enlarged to allow the annotation of skin structures.

Mentions: While the skin surface localization patterns delineate the global expression trends, skin is a squamous epithelial tissue so it was possible that cells below the observable surface (and so not apparent on the surface view) were also being transfected. Through the natural migration of cells in the epidermis, such cells would only later move to the skin surface and so give a falsely slow dynamic. To investigate this further, biopsies were taken of the time points from the original study and fixed sections prepared. Under a fluorescent microscope, sections were observed and scored for GFP positive cells (Figure 2). One hour post treatment, positive cells were identified in skin sections. These cells appeared to be closer to the basement membrane and were likely to be located in the stratum basale level of the epidermis and the mid to upper epidermis (stratum granulosum). As the time course progressed, the transfected cells migrated upwards to the surface of the skin where between day 5 and day 7 the majority of the signal appeared to reside in the stratum corneum. The seemingly brighter GFP signal at day 7 over day 3 is a function of the change in morphology of the reporter gene positive calls as they flatten out in the SC. Post day 7, the GFP positive cells appeared to slough off which is the nature of that barrier layer of the skin. To allow annotation of the structural elements in the skin sections, an enlarged view of the 2 h section is included where the sub-structures and stratum of the skin are noted. In keeping with the shallow electrical field generated by this EP device, all the transfected cells were localized in the epidermis of the skin sections.


Elucidating the Kinetics of Expression and Immune Cell Infiltration Resulting from Plasmid Gene Delivery Enhanced by Surface Dermal Electroporation.

Mendoza JM, Amante DH, Kichaev G, Knott CL, Kiosses WB, Smith TR, Sardesai NY, Broderick KE - Vaccines (Basel) (2013)

Histological analysis reveals rapid GFP expression. Histological analysis of GFP expression in a time course (1-hour post treatment to day 21) after ID plasmid administration followed by EP with SEP in guinea pig skin. Skin biopsies were removed, cryosectioned, DAPI stained and visualized under fluorescence microscopy (10×). An injection only control is also shown. Photos are representative examples of multiple treatments. A region of the 2-hour time point section image is enlarged to allow the annotation of skin structures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494224&req=5

vaccines-01-00384-f002: Histological analysis reveals rapid GFP expression. Histological analysis of GFP expression in a time course (1-hour post treatment to day 21) after ID plasmid administration followed by EP with SEP in guinea pig skin. Skin biopsies were removed, cryosectioned, DAPI stained and visualized under fluorescence microscopy (10×). An injection only control is also shown. Photos are representative examples of multiple treatments. A region of the 2-hour time point section image is enlarged to allow the annotation of skin structures.
Mentions: While the skin surface localization patterns delineate the global expression trends, skin is a squamous epithelial tissue so it was possible that cells below the observable surface (and so not apparent on the surface view) were also being transfected. Through the natural migration of cells in the epidermis, such cells would only later move to the skin surface and so give a falsely slow dynamic. To investigate this further, biopsies were taken of the time points from the original study and fixed sections prepared. Under a fluorescent microscope, sections were observed and scored for GFP positive cells (Figure 2). One hour post treatment, positive cells were identified in skin sections. These cells appeared to be closer to the basement membrane and were likely to be located in the stratum basale level of the epidermis and the mid to upper epidermis (stratum granulosum). As the time course progressed, the transfected cells migrated upwards to the surface of the skin where between day 5 and day 7 the majority of the signal appeared to reside in the stratum corneum. The seemingly brighter GFP signal at day 7 over day 3 is a function of the change in morphology of the reporter gene positive calls as they flatten out in the SC. Post day 7, the GFP positive cells appeared to slough off which is the nature of that barrier layer of the skin. To allow annotation of the structural elements in the skin sections, an enlarged view of the 2 h section is included where the sub-structures and stratum of the skin are noted. In keeping with the shallow electrical field generated by this EP device, all the transfected cells were localized in the epidermis of the skin sections.

Bottom Line: Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen.In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space.These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

View Article: PubMed Central - PubMed

Affiliation: Inovio Pharmaceuticals Inc., 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422, USA.

ABSTRACT
The skin is an attractive tissue for vaccination in a clinical setting due to the accessibility of the target, the ease of monitoring and most importantly the immune competent nature of the dermal tissue. While skin electroporation offers an exciting and novel future methodology for the delivery of DNA vaccines in the clinic, little is known about the actual mechanism of the approach and the elucidation of the resulting immune responses. To further understand the mechanism of this platform, the expression kinetics and localization of a reporter plasmid delivered via a surface dermal electroporation (SEP) device as well as the effect that this treatment would have on the resident immune cells in that tissue was investigated. Initially a time course (day 0 to day 21) of enhanced gene delivery with electroporation (EP) was performed to observe the localization of green fluorescent protein (GFP) expression and the kinetics of its appearance as well as clearance. Using gross imaging, GFP expression was not detected on the surface of the skin until 8 h post treatment. However, histological analysis by fluorescent microscopy revealed GFP positive cells as early as 1 h after plasmid delivery and electroporation. Peak GFP expression was observed at 24 h and the expression was maintained in skin for up to seven days. Using an antibody specific for a keratinocyte cell surface marker, reporter gene positive keratinocytes in the epidermis were identified. H&E staining of treated skin sections demonstrated an influx of monocytes and granulocytes at the EP site starting at 4 h and persisting up to day 14 post treatment. Immunological staining revealed a significant migration of lymphocytic cells to the EP site, congregating around cells expressing the delivered antigen. In conclusion, this study provides insights into the expression kinetics following EP enhanced DNA delivery targeting the dermal space. These findings may have implications in the future to design efficient DNA vaccination strategies for the clinic.

No MeSH data available.