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Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin

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Abstract

Purpose

Small interfering RNAs (siRNAs) specifically and potently inhibit target gene expression. Pachyonychia congenita (PC) is a skin disorder caused by mutations in genes encoding keratin (K) 6a/b, K16, and K17, resulting in faulty intermediate filaments. A siRNA targeting a single nucleotide, PC-relevant mutation inhibits K6a expression and has been evaluated in the clinic with encouraging results.

Procedures

To better understand the pathophysiology of PC, and develop a model system to study siRNA delivery and visualize efficacy in skin, wild type (WT) and mutant K6a complementary DNAs (cDNAs) were fused to either enhanced green fluorescent protein or tandem tomato fluorescent protein cDNA to allow covisualization of mutant and WT K6a expression in mouse footpad skin using a dual fluorescence in vivo confocal imaging system equipped with 488 and 532 nm lasers.

Results

Expression of mutant K6a/reporter resulted in visualization of keratin aggregates, while expression of WT K6a/reporter led to incorporation into filaments. Addition of mutant K6a-specific siRNA resulted in inhibition of mutant, but not WT, K6a/reporter expression.

Conclusions

Intravital imaging offers subcellular resolution for tracking functional activity of siRNA in real time and enables detailed analyses of therapeutic effects in individual mice to facilitate development of nucleic acid-based therapeutics for skin disorders.

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Acknowledgments

The authors would like to thank Conor Cox for his efforts in acquiring the confocal microscopy and fluorescent histological data. We also thank Robert Kaspar, Heini Ilves, and Jed Humphries for technical support and Andrea Burgon for administrative support. This work was supported by NIH grants R44AR056559 (RLK, CHC) and the Chambers Family Foundation (CHC).

Conflict of Interest

Roger Kaspar and Robyn Hickerson have patents issued and pending on using siRNA to treat PC and siRNA delivery technologies.

Author information

Author notes

  1. Robyn P. Hickerson

    Present address: Centre for Dermatology and Genetic Medicine, University of Dundee, Dundee, UK

  2. Maria Fernanda Lara

    Present address: Urology Research Unit Virgen de la Victoria and Regional Hospital, Malaga, Spain

  3. Emilio González-González

    Present address: Canvax Biotech S.L., Technological Park, Cordoba, Spain

Authors and Affiliations

  1. TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA

    Robyn P. Hickerson, Tycho J. Speaker, Maria Fernanda Lara, Manuel A. Flores & Roger L. Kaspar

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

    Emilio González-González & Christopher H. Contag

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

    Emilio González-González & Christopher H. Contag

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

    Christopher H. Contag

  5. Departments of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Christopher H. Contag

Authors
  1. Robyn P. Hickerson
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  2. Tycho J. Speaker
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  3. Maria Fernanda Lara
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  4. Emilio González-González
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  5. Manuel A. Flores
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  6. Christopher H. Contag
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  7. Roger L. Kaspar
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Corresponding author

Correspondence to Roger L. Kaspar.

Electronic Supplementary Material

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Supplementary Video 1

(Movie showing full dataset from Fig. 3.) Separate intradermal injections of distinct reporter expression plasmids to mouse footpads results in distinct subsets of keratinocytes that express each reporter protein. To determine if successive intradermal injections would result in delivery and expression of reporter genes in the same, or different, subsets of cells, two separate injections (12 h apart) of plasmids expressing differentially-labeled reporter proteins were performed. The expression construct pUBC-K6a(N171K)-tdTFP was injected into a mouse footpad at time 0. At 12 h, pUBC-K6a(WT)-EFGP was injected into the same footpad, as close to the original injection site as possible. The footpad skin was imaged at 36 h using the intravital scope. a Reflectance was used to visualize skin structure and create a reference image. Fluorescence using b 488 and c 532 nm lasers are d overlaid to show the subsets of cells expressing the reporter protein from each injection. (AVI 10221 kb)

Supplementary Video 2

As described in Fig. 4, mouse paws were imaged with the confocal imaging VivaScope. Movie shows all images collected every 2 μm from the skin surface to a depth of 130 μm. En face images using reflectance and green and red fluorescence from both paws are shown. Note that the left panel in (c) is reflectance microscopy to show general skin structure. (AVI 32240 kb)

Supplementary Video 3

(Movie showing full dataset from Fig. 5.) Intravital visualization of mutant and wildtype K6a expression in skin. The expression plasmids pUBC-K6a(WT)-EGFP (pTD239, a) or pUBC-K6a(N171K)-EGFP (pTD240, b) were intradermally injected (40 μg in 70 μL PBS) into mouse footpads and imaged (24 h). a WT K6a expression is generally uniformly expressed throughout the cytoplasm of each cell leaving a dark nuclear region in the center of each cell (arrows depict examples). b Mutant N171K K6a/EGFP expression results in pronounced (as compared to WT expression) aggregation. (AVI 3714 kb)

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Hickerson, R.P., Speaker, T.J., Lara, M.F. et al. Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin. Mol Imaging Biol 18, 34–42 (2016). https://doi.org/10.1007/s11307-015-0875-z

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  • DOI: https://doi.org/10.1007/s11307-015-0875-z

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