title: Non-Invasive Intravital Imaging of siRNA-Mediated Mutant Keratin Gene Repression in Skin.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
authors.affiliation: TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA.|Centre for Dermatology and Genetic Medicine, University of Dundee, Dundee, UK.
authors.forename: Robyn P
authors.initials: RP
authors.lastname: Hickerson
authors.affiliation: TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA.
authors.forename: Tycho J
authors.initials: TJ
authors.lastname: Speaker
authors.affiliation: TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA.|Urology Research Unit Virgen de la Victoria and Regional Hospital, Malaga, Spain.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Lara
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA, USA.|Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.|Canvax Biotech S.L., Technological Park, Cordoba, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: González-González
authors.affiliation: TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA.
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Flores
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA, USA.|Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.|Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA.|Departments of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Contag
authors.affiliation: TransDerm Inc., 2161 Delaware Ave., Santa Cruz, CA, 95060, USA. roger.kaspar@transderminc.com.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Kaspar
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007271:Injections, Intradermal; D007633:Keratins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D066329:Protein Aggregates; D034741:RNA, Small Interfering / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D066329:Protein Aggregates; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007633:Keratins
keywords: Gene regulation; Gene therapy; Genodermatosis; In vivo confocal fluorescence microscopy