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Anna Paola Salvetti, Maria Ines Patricio, Alun R Barnard, Robert Maclaren; Impact of vital dyes on transduction efficiency of AAV vectors used in retinal gene therapy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):104.
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© 2017 Association for Research in Vision and Ophthalmology.
Treatment of inherited retinal degenerations (IRD) using adeno-associated viral (AAV) vectors typically involves delivery of the viral vector under the macula by sub-retinal injection through a very small retinotomy. In the late stages of IRD a progressive alteration of the normal anatomy can cause difficulties in localizing the retinotomy, the limit of the retinal detachment (RD) and the AAV vector diffusion. Vital dyes can be a useful surgical adjuvant in routine vitreoretinal surgery, particularly for detecting the internal limiting membrane. The impact of vital dyes on AAV transduction is, however, unknown.
Different dilutions of two dyes, Sodium Fluorescein (SF, Martindale Pharma, UK) and Membrane Blue Dual (MBD, D.O.R.C., The Netherlands), were tested on Human Embryonic Kidney cells (HEK293) in vitro using an AAV2.GFP (green fluorescent protein) reporter. Flow Cytometer (FC) analysis was performed to assess both HEK 293 cell viability and AAV2.GFP transduction efficiency in triplicate. A Live/Dead Violet Fixable Dead Cell Stain was added to differentiate live and dead cells.A range of SF (1:0, 1:50, 1:5.000, 1:50.000 and 1:500.000) and MBD (1:0, 1:50, 1:125, 1:150 and 1:500) dilutions in tissue culture media were tested. Cells were afterwards incubated in order to allow AAV2.GFP transduction for three further days, FC was performed and data collected.
The potential toxicity of the range of MBD and SF dilutions was compared with the negative control. The positive control was composed by dead cells killed by heating. MBD and SF were toxic only when applied undiluted to HEK293 cells. For clinical applications, 1:5.000 for SF and 1:125 for MBD were the dilutions that remained clearly visible to the human eye with a potential use in surgery. AAV2.GFP transduction efficiency at these dilutions was tested and was not reduced by the presence of the dyes when compared to the non-dye control.
Cells viability and transduction efficiency were not affected by the presence of the dyes when diluted, as would be in the case in vitreo-retinal surgery. SF and MBD may therefore be a safe adjuvant during surgery to enable a better visualization of the retina and of the vector diffusion during gene therapy surgery.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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