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

遗传性视网膜营养不良症（IRDs）包括一组不同的致盲疾病，导致进行性视网膜丧失。目前，大多数治疗方法集中于单个基因或突变。一个更有吸引力的替代方案是开发能够独立于潜在基因或疾病机制阻止疾病进展的策略。表达神经保护基因或敲除潜在有害基因是实现这一目标的一种有希望的方法。过去已经发现了几种神经保护或细胞损伤基因或蛋白质，并且对其中一些基因或蛋白质的补充或敲除已经在小鼠模型中进行了评估。然而，尽管取得了一些进展，但尚不清楚不同的细胞保护蛋白及其受体的组合或同时敲除潜在的破坏性基因是否对治疗成功的效率和持续时间有额外的有益影响。为了能够在体内测试这种组合方法，我们将应用模块化CRISPR-Cas技术，该技术适用于使用位点特异性单导rna （sgRNAs）激活单个或多个基因（CRISPRa）。在前期工作中，我们已经成功地将CRISPRa应用于IRD小鼠模型的基因治疗。在进一步的前期工作中，我们已经证明CRISPRa也适合在体内同时敲低其他基因。在拟议的实验中，我们将首先在体外优化适合激活编码神经保护蛋白及其受体的基因（transactivation multiplexing unit， TAMU）的不同sgrna。在进一步的实验中，我们将在体外和体内测试不同TAMU组合在RNA和蛋白质水平上的效率。在体内应用中，TAMUs和CRISPRa将使用双重组腺相关病毒（rAAV）载体进行表达。利用Pde6bH620Q/Cre小鼠色素性视网膜炎模型分析优化后TAMUs的效率。Pde6bH620Q/Cre小鼠的优点是，注射他莫昔芬可以在任何时间点停止退行性过程，从而可以测量要达到的最大治疗效果。将在注射后的不同时间点从分子、形态、功能和行为水平评估个体TAMUs的治疗成功，并与注射他莫昔芬的小鼠进行比较。最有效的TAMU将与敲低的sgrna结合，并在Pde6bH620Q/Cre小鼠模型中进行评估。如果成功，这种方法将为不依赖基因的组合基因治疗提供原理证明，这将使全世界数百万患者受益。

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.