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

TY - JOUR

T1 - Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin

AU - Hickerson, Robyn P.

AU - Speaker, Tycho J.

AU - Lara, Maria Fernanda

AU - González-González, Emilio

AU - Flores, Manuel A.

AU - Contag, Christopher H.

AU - Kaspar, Roger L.

N1 - This work was supported by NIH grants R44AR056559 (RLK, CHC) and the Chambers Family Foundation (CHC).

PY - 2016/2

Y1 - 2016/2

N2 - 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.

AB - 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.

KW - Gene regulation

KW - Gene therapy

KW - Genodermatosis

KW - In vivo confocal fluorescence microscopy

UR - https://www.scopus.com/pages/publications/84955492918

U2 - 10.1007/s11307-015-0875-z

DO - 10.1007/s11307-015-0875-z

M3 - Article

C2 - 26169581

AN - SCOPUS:84955492918

SN - 1536-1632

VL - 18

SP - 34

EP - 42

JO - Molecular Imaging and Biology

JF - Molecular Imaging and Biology

IS - 1

ER -