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

用于视网膜变性的基因独立治疗的多重 CRISPRa 方法

• 批准号: 399299380
• 负责人: Professor Dr. Martin Biel, since 6/2022
• 金额: --
• 依托单位: Department Pharmazie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/399299380?language=en
• 关键词: multiplexing; CRISPRa; approach; gene; independent

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.

遗传性视网膜营养不良（IRD）包括导致进行性视网膜丧失的一组广泛的不同致盲疾病。目前，大多数治疗方法集中在单个基因或突变。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。神经保护基因的表达或潜在有害基因的敲除代表了实现这一目标的有希望的方法。过去已经鉴定了几种神经保护或细胞损伤基因或蛋白质，并且对于其中一些，已经在小鼠模型中评估了它们的补充或敲除的效果。然而，尽管取得了一些进展，但仍不清楚不同细胞保护蛋白及其受体的组合或潜在破坏性基因的同时敲除是否对治疗成功的效率和持续时间具有额外的有益影响。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，该技术适用于使用基因座特异性单向导RNA（sgRNA）激活单个或多个基因（CRISPRa）。在我们的初步工作中，我们已经成功地将CRISPRa应用于IRD小鼠模型的基因治疗。在进一步的初步工作中，我们已经证明CRISPRa也适合于在体内同时敲除其他基因。在所提出的实验中，我们将首先在体外优化适合于激活编码神经保护蛋白及其受体的基因的不同sgRNA（反式激活多路复用单元，TAMU）。在进一步的实验中，我们将在体外和体内在RNA和蛋白质水平上测试不同TAMU组合的效率。对于体内应用，将使用双重重组腺相关病毒（rAAV）载体表达TAMU和CRISPRa。优化的TAMU的效率将使用视网膜色素变性的Pde 6 bH 620 Q/Cre小鼠模型进行分析。Pde 6 bH 620 Q/Cre小鼠提供的优点是，可以在任何时间点通过他莫昔芬注射停止退行性过程，因此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在分子、形态、功能和行为水平上评估单个TAMU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组合，并在Pde 6 bH 620 Q/Cre小鼠模型中进行评估。如果成功，这种方法将为基因独立的组合基因治疗提供原理证明，可以使全球数百万患者受益。