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R. L. Muehlfriedel, M. D. Fischer, S. Michalakis, N. Tanimoto, G. Huber, S. C. Beck, E. Bécirovi, M. Biel, M. W. Seeliger; Acute Phase and Long-Term Effects of Subretinal Gene Transfer in Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3126.
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© ARVO (1962-2015); The Authors (2016-present)
To follow morphological effects of retinal gene transfer associated with subretinal injections on retinal layer integrity in mice.
Retinal morphology of injected and untouched eyes was analyzed using optical coherence tomography (SD-OCT) and confocal scanning-laser ophthalmoscopy (SLO) immediately upon completion of subretinal injections, and at different subsequent time points (from 3 days to 16 months post treatment). Transfected areas were identified by local EGFP expression under the transcriptional control of CMV, Arrestin3, MWS / RG, SWS, and Rho promoters. Results were confirmed by immunohistochemistry and retinal whole mounts.
To confirm the success of injection and to enable a later correlation with the transduced area, the same individual animals were examined sequentially with SLO and OCT imaging. Initially, a defined local retinal detachment was present immediately after subretinal injection. This detachment resolved quickly within the first week post injection. With the exception of a scar at the site of injection, the procedure did in most cases not leave any perceivable traces behind (also electrophysiologically). Later on, the respective area could be best visualized in autofluorescence mode due to EGFP expression, which correlated well to the initial bleb size. Furthermore, differences in the latency period of EGFP expression are observed among different vector systems (AAV vs. lentivirus) and promoters. A terminal analysis of retinal whole mounts revealed that approximately 1/3 to 1/4 of the retinal area was transduced by a single injection.
Little efforts have been made so far to study and potentially optimize injection conditions in gene therapy. Here, we show for the first time a conclusive series of sequential examinations initially focused on size and resorption time of the formed cavity, and later on morphological integrity (assessed with OCT) and transduction yield (assessed by SLO autofluorescence). The non-invasive detection of expression markers like EGFP provides important information for the optimization of the therapeutic efficacy and specificity of gene therapy approaches.
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