In photodegenerative diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD),progressive loss of vision occurs as a result of degeneration of the periphery of the retina and the macula,respe...In photodegenerative diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD),progressive loss of vision occurs as a result of degeneration of the periphery of the retina and the macula,respectively.Current optogenetic stimulation-based approaches to vision restoration offer the advantages of cellular specificity,high resolution,and minimal invasiveness over electrode arrays;however,the clinical translation of optogenetic activation suffers from the lack of a method for the delivery of opsins into spatially targeted regions of a retina that has degenerated.Non-targeted opsin delivery through viral or non-viral methods to non-photodegenerated retinal areas will perturb these already functioning retinal regions.Furthermore,viral methods are subject to limitations on the delivery of large plasmids,such as fusion constructs of multiple spectrally separated opsins(e.g.,channelrhodopsin-2(ChR2),chimeric opsin variants(C1V1),ReaChR),which can provide higher photo-excitability than can a single narrow-band opsin under ambient light conditions.Here,we report the ultrafast near-infrared laser-based spatially targeted transfection of single and multiple opsins and present a comparison with the opsin expression distribution achieved using another non-viral,but non-targeted,transfection method,lipofection.Functional evaluation of cells transfected with multiple opsins using the laser method revealed a significantly higher white-light-induced photocurrent than in cells expressing a single opsin(ChR2).The laser-assisted targeted delivery of multiple opsin-encoding genes to the peripheral retina/macula is ideal for sensitizing retinal areas that have degenerated,thus paving the way toward the restoration of lost vision in RP/AMD patients.展开更多
基金the support from the National Institute of Health(NS084311)the Office of the President and Provost of The University of Texas at Arlington.
文摘In photodegenerative diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD),progressive loss of vision occurs as a result of degeneration of the periphery of the retina and the macula,respectively.Current optogenetic stimulation-based approaches to vision restoration offer the advantages of cellular specificity,high resolution,and minimal invasiveness over electrode arrays;however,the clinical translation of optogenetic activation suffers from the lack of a method for the delivery of opsins into spatially targeted regions of a retina that has degenerated.Non-targeted opsin delivery through viral or non-viral methods to non-photodegenerated retinal areas will perturb these already functioning retinal regions.Furthermore,viral methods are subject to limitations on the delivery of large plasmids,such as fusion constructs of multiple spectrally separated opsins(e.g.,channelrhodopsin-2(ChR2),chimeric opsin variants(C1V1),ReaChR),which can provide higher photo-excitability than can a single narrow-band opsin under ambient light conditions.Here,we report the ultrafast near-infrared laser-based spatially targeted transfection of single and multiple opsins and present a comparison with the opsin expression distribution achieved using another non-viral,but non-targeted,transfection method,lipofection.Functional evaluation of cells transfected with multiple opsins using the laser method revealed a significantly higher white-light-induced photocurrent than in cells expressing a single opsin(ChR2).The laser-assisted targeted delivery of multiple opsin-encoding genes to the peripheral retina/macula is ideal for sensitizing retinal areas that have degenerated,thus paving the way toward the restoration of lost vision in RP/AMD patients.
