title: Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase.
abstract: Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10-14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.
authors.affiliation: Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Division of Environmental Dermatology and Allergy, National Research Center for Environment & Health, Technische Universität München and GSF, Munich, Germany. mbf@lrz.tum.de
authors.forename: Markus
authors.initials: M
authors.lastname: Braun-Falco
authors.forename: Angelika
authors.initials: A
authors.lastname: Eisenried
authors.forename: Hildegard
authors.initials: H
authors.lastname: Büning
authors.forename: Johannes
authors.initials: J
authors.lastname: Ring
mesh_terms: D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D000229:Dependovirus / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D012871:Skin Diseases / Q000628:therapy*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde