siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model

Abstract

Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/ humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

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

“…injection of unmodified and unformulated siRNA into the footpad of a transgenic skin reporter mouse. 35 Furthermore, in the recently reported pachyonychia congenita phase 1b clinical trial, very large volumes (up to two ml) were intradermally injected and high pressure may have been responsible for the observed effect on keratin gene expression. 7 Although i.d.…”

“…Fluorescence was predominantly localized in the upper layers of the epidermis, similar to what we previously reported in a hMGFP transgenic mouse model. 35 The strong fluorescence signal of this reporter construct allowed clear visualization of single keratinocytes expressing hMGFP as early as 6 h (Figures 3a and b) after i.d. injection (in vivo detection of fluorescence using the DAC intravital imaging system was difficult due to low hMGFP signals at these early time points).…”

“…delivery of hMGFP reporter (driven by the CMV promoter) in the upper layers of the epidermis mirror the pattern observed in a tgCBL/hMGFP transgenic mouse skin model that we previously reported. 35 This mouse expresses hMGFP under the control of the chick b-actin (CAG) promoter (Supplementary Figures 1a, b see also Gonzalez-Gonzalez 35 ), which also contains CMV enhancer elements. A similar suprabasal pattern of reporter expression has been observed following intradermal plasmid injections 38,[51][52][53] as well as in transgenic animals, 54,55 where transcription is driven by CMV or CAG promoters.…”

“…The pCMV-hMGFP/CBL plasmid produces a fusion protein consisting of humanized Montastrea cavernosa ('monster') green fluorescence reporter protein followed by click beetle luciferase (CBL), under the control of the CMV promoter. 35 The CMV promoter in pCMV-hMGFP/CBL was replaced with the following promoters: eIF4A1, Ubc and EF1a (TD153, TD154 and TD151, respectively). All enzymes were purchased from New England Biolabs (Ipswich, MA, USA).…”

“…We have recently reported that the DAC microscope can readily detect siRNA-mediated inhibition of green fluorescent protein (GFP) reporter expression in a transgenic GFP mouse model. 35 To investigate the utility of intravital imaging to monitor delivery of nucleic acids to skin, and to improve our understanding of the mechanisms, including high pressure, involved in nucleic acid uptake and functionality (that is, gene expression) in cells of the epidermis, we used fluorescence microscopy (including intravital DAC imaging) and in vivo bioluminescence imaging (BLI) to monitor the transfer of plasmid DNA and expression of encoded genes following i.d. injection.…”