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Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters

Gene Therapy volume 17, pages 1270–1278 (2010)Cite this article

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Abstract

Nucleic acid-based therapies hold great promise for treatment of skin disorders if delivery challenges can be overcome. To investigate one mechanism of nucleic acid delivery to keratinocytes, a fixed mass of expression plasmid was intradermally injected into mouse footpads in different volumes, and reporter expression was monitored by intravital imaging or skin sectioning. Reporter gene expression increased with higher delivery volumes, suggesting that pressure drives nucleic acid uptake into cells after intradermal injections similar to previously published studies for muscle and liver. For spatiotemporal analysis of reporter gene expression, a dual-axis confocal (DAC) fluorescence microscope was used for intravital imaging following intradermal injections. Individual keratinocytes expressing hMGFP were readily visualized in vivo and initially appeared to preferentially express in the stratum granulosum and subsequently migrate to the stratum corneum over time. Fluorescence microscopy of frozen skin sections confirmed the patterns observed by intravital imaging. Intravital imaging with the DAC microscope is a noninvasive method for probing spatiotemporal control of gene expression and should facilitate development and testing of new nucleic acid delivery technologies.

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Abbreviations

DAC:

dual-axis confocal

CBL:

click beetle luciferase

hMGFP:

humanized monster green fluorescent protein

siRNA:

small-interfering RNA

RNAi:

RNA interference

Ubc:

ubiquitin C

eIF4A1:

eukaryotic translation initiation factor 4A, isoform 1

EF1a:

elongation factor 1a

BLI:

bioluminescence imaging

i.d.:

intradermal

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Acknowledgements

We thank Manny Flores and Heini Ilves for technical support, Tycho Speaker for assistance in data analysis, Cory Nicholas (Stanford University) for providing C57BL/6-Tg(UBC-GFP)30Scha/J and B6;129S-Gt(ROSA)26Sor/J transgenic animals, Jonathan Hardy, Thomas Kledal and Michael Bachmann for providing pEIF4A1 and pTEJ-8 and Soosan Ghazizadeh for providing pHR′EF1–GFP–WPRESIN18 and for useful discussion. This work was supported by NIH Grants R43AR055881-01 and R44AR055881-02 (RLK); and U54 CA105296 (CHC). Emilio Gonzalez is the recipient of a Pachyonychia Congenita Project fellowship.

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Authors and Affiliations

  1. Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA, USA

    E González-González, H Ra, R Spitler & C H Contag

  2. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    E González-González, H Ra, R Spitler & C H Contag

  3. Department of Electrical Engineering, Stanford University, Stanford, CA, USA

    H Ra

  4. TransDerm Inc.,, Santa Cruz, CA, USA

    R Spitler, R P Hickerson & R L Kaspar

  5. Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA

    C H Contag

  6. Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, USA

    C H Contag

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Correspondence to R L Kaspar.

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Competing interests

Roger Kaspar, Robyn Hickerson and Ryan Spitler are employees of TransDerm Inc. and Christopher Contag is a founder of Xenogen Corp. now part of Caliper LifeSciences.

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González-González, E., Ra, H., Spitler, R. et al. Increased interstitial pressure improves nucleic acid delivery to skin enabling a comparative analysis of constitutive promoters. Gene Ther 17, 1270–1278 (2010). https://doi.org/10.1038/gt.2010.74

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