Efficacy of gene transfer through the round window membrane: an in vitro model.
The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.
Department of ORL, HUCH, University of Helsinki, Helsinki, Finland. antti.aarnisalo@hus.fi
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Aarnisalo
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Aarnisalo
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Pietola
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Wahlfors
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Jero
D000818:Animals; D003051:Cochlea / Q000378:metabolism*; D004396:Coloring Agents; D005781:Gelatin Sponge, Absorbable / Q000378:metabolism; D018014:Gene Transfer Techniques; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D066298:In Vitro Techniques; D049408:Luminescent Agents; D008297:Male; D051379:Mice; D023421:Models, Animal; D010539:Permeability; D012405:Round Window, Ear / Q000378:metabolism*; D014048:Tolonium Chloride
D004396:Coloring Agents; D005819:Genetic Markers; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; D014048:Tolonium Chloride