Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

Lars Steinstraesser,Jadwiga Stupka,Tobias Hirsch,Frank Jacobsen,Andreas David Niederbichler,Jan-Michel Otte,Matthias Schulte,Sammy Al-Benna,Marco Rainer Kesting,Hans-Ulrich Steinau,Michael Sorkin

doi:10.1186/1471-2172-12-8

Abstract

BackgroundAdenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes.MethodsIn vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector.ResultsThe results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo.ConclusionThe results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

Highlights

Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications
desoxyribonucleic acid (DNA) internalization induced innate immune reaction In order to get a deeper insight into the molecular mechanisms of the innate immune system after DNA internalization into keratinocytes, we decided to use a green fluorescent protein (GFP)-encoding adenovirus for this study
Since this generation of adenoviral vectors exhibit a high immunogenicity, this vector seemed to be an interesting tool for the investigation of signal transduction moleculess involved in adenovirus detection

Summary

Introduction

Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. These vectors can induce immune responses that may severely affect the ability of vector reapplication. The skin consists of three main layers - the epidermis, the dermis, and the hypodermis, each with their own function. It is the epidermis, which creates a barrier to and protects from pathogens of the outside world. This highly specialised layer is mainly composed of keratinocytes, melanocytes and dendritic cells. As the epidermis is known to produce a variety of cytokines and growth factors, keratinocytes may be engineered as bioreactors to secrete gene products which have local or systemic effects [4,5]

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