A multiplexing CRISPRa approach for gene-independent therapy of retinal degeneration

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Inherited retinal dystrophies (IRDs) comprise a broad group of different blinding disorders that result in progressive retinal loss. Currently, most therapeutic approaches focus on single genes or mutations. A much more attractive alternative is to develop strategies that can halt disease progression independent of the underlying gene or disease mechanism. Expression of neuroprotective genes or knockdown of potentially harmful genes represents a promising way to achieve this goal. Several neuroprotective or cell-damaging genes or proteins have been identified in the past, and for some of them, the effect of their supplementation or knockdown has been evaluated in mouse models. However, despite some progress, it remains unclear whether a combination of different cell-protective proteins and their receptors or the simultaneous knockdown of potentially damaging genes have additional beneficial effects on the efficiency and duration of therapeutic success. To enable the testing of such combinatorial approaches in vivo, we will apply the modular CRISPR-Cas technology, which is suitable for activating single or multiple genes (CRISPRa) using locus-specific single guide RNAs (sgRNAs). In our preliminary work, we have already successfully applied CRISPRa for gene therapy of an IRD mouse model. In further preliminary work, we have shown that CRISPRa is also suitable for simultaneous knockdown of other genes in vivo. In the proposed experiments, we will first optimize different sgRNAs in vitro that are suitable for activating genes encoding neuroprotective proteins and their receptors (transactivation multiplexing unit, TAMU). In further experiments, we will test the efficiency of different TAMU combinations at the RNA and protein levels in vitro and in vivo. For in vivo application, the TAMUs and CRISPRa will be expressed using dual recombinant adeno-associated viral (rAAV) vectors. The efficiency of optimized TAMUs will be analyzed using the Pde6bH620Q/Cre mouse model for retinitis pigmentosa. Pde6bH620Q/Cre mice offer the advantage that the degenerative process can be stopped by tamoxifen injection at any time point and thus the maximal therapeutic effect to be achieved can be measured. The therapeutic success of individual TAMUs will be assessed at various time points after injection at the molecular, morphological, functional and behavioral levels and compared to tamoxifen injected mice. The most efficient TAMU will be combined with knockdown sgRNAs and evaluated in the Pde6bH620Q/Cre mouse model. If successful, this approach will provide a proof-of-principle for gene-independent combinatorial gene therapy that could benefit millions of patients worldwide.