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W. H. Merigan, D. Scoles, J. J. Hunter, B. Masella, K. P. Greenberg, J. G. Flannery, R. T. Libby, D. R. Williams; Tracking Transfection of Macaque Retinal Ganglion Cells With AAV2 Viral Vectors; In vivo Imaging Reveals Differences Between Two Promoters. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4514. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a method for viral mediated gene transfer to primate retinal ganglion cells using in vivo imaging and histological analysis to track the selectivity of transfection.
AAV2 viral vectors containing either chicken beta-actin or connexin (CX36) promoters driving GFP were injected intravitreally into macaque eyes. The degree of transfection was monitored in vivo with adaptive optics imaging and histologically by microscopy
We observed a prolonged delay of several months between intravitreal injection of the viral particles and visible GFP expression for vectors containing the chicken beta-actin promoter. GFP expression was first observed in foveal cones at 43 weeks, and only at 57 weeks was GFP visible in an annulus of densely labelled retinal ganglion cells (RGCs) around the fovea. Histological analysis indicated additional labeling in the periphery of the retina that was not visible with in vivo imaging, especially sparse labeling of presumptive Muller cells and RGCs. On the other hand, AAV vectors of the same serotype and titer containing the connexin promoter produced rapid onset (2 weeks) and dense labeling of circumfoveal RGCs. This promoter did not produce transfection of cone photoreceptors. In addition, GFP labeling of peripheral RGCs, small numbers of Muller cells and some cells in the inner nuclear layer were found on microscopy.
AAV2 viral vectors containing two different promoters both produced a striking pattern of dense labeling of RGCs in an annulus around the fovea. However, results with the two promoters differed in that only the beta-actin promoter produced labeling of foveal cones, and only the connexin promoter produced a rapid appearance of GFP in retinal neurons. Transfection of primate RGCs combined with in vivo imaging at multiple timepoints will be useful for a variety of studies relevant to human diseases affecting ganglion cells as well as neuroscience investigations of visual processing.
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