RESUMO
Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration and loss of light-sensing photoreceptors. The most promising therapeutic approach for IRDs is gene supplementation therapy using viral vectors, which requires the presence of viable photoreceptors at the time of intervention. At later disease stages, photoreceptors are lost and can no longer be rescued with this approach. For these patients, conferring light-sensing abilities to the remaining interneurons of the ON circuit (i.e., ON bipolar cells) using optogenetic tools poses an alternative treatment strategy. Such treatments, however, are hampered by the lack of efficient gene delivery tools targeting ON bipolar cells, which in turn rely on the effective isolation of these cells to facilitate tool development. Herein, we describe a method to selectively isolate ON bipolar cells via fluorescence-activated cell sorting (FACS), based on the expression of two intracellular markers. We show that the method is compatible with highly sensitive downstream analyses and suitable for the isolation of ON bipolar cells from healthy as well as degenerated mouse retinas. Moreover, we demonstrate that this approach works effectively using non-human primate (NHP) retinal tissue, thereby offering a reliable pipeline for universal screening strategies that do not require inter-species adaptations or transgenic animals.
RESUMO
Curing a genetic disease by repairing the underlying genetic defect is a fascinating concept that has been addressed so far by gene compensation therapy. For this, a functional copy of the gene in question together with elements controlling its expression is produced as a vector and introduced ex vivo into the patient's own cells that subsequently are reinfused. Alternatively, vectors are administered directly in vivo. Although this strategy resulted in impressive therapeutic benefits for patients, the ultimate goal of gene therapy, i.e., a cure by repairing the actual genetic or epigenetic defect, remained an unresolved task. With the advent of designer DNA-binding domains, this goal is coming into reach. These domains are either combined with nucleases and used as molecular precision scissors for introducing DNA breaks at defined sites in the cell's genome preparing for position-selective DNA repair, or they are used as programmable DNA-binding units for positioning epigenome-modifying domains to predefined target sequences. However, for reaching its full potential, these components need to be delivered into cells in an efficient and safe manner. Here, we summarize current viral and non-viral delivery approaches applicable for genome and epigenome editing and discuss their respective advantages and limitations.