Pathway engineering to optimize homology dependent therapeutic genome editing at the ABCA4 locus in photoreceptors

200多个视网膜特异基因的突变与遗传性视网膜疾病(IRD)有关。基于基因的治疗，主要是以基因添加的形式或使用腺相关病毒(AAV)载体进行基因传递的补充治疗，已经针对几种IRD进行了开发。然而，到目前为止，所有的靶向疾病都与足够小的基因突变有关，以至于相应的cdna可以在一个AAV中转移。不幸的是，许多最频繁的突变基因，如ABCA4，都超过了AAV的货物大小。ABCA4突变导致Stargardt病，这是一种早期发病的黄斑变性形式。由于ABCA4序列长度的原因，Stargardt病的基因治疗受到阻碍，基因编辑是纠正患者光感受器基因组突变的一种有吸引力的方法。精确的基因编辑，以避免不想要的和不受控制的额外基因组改变，需要同源依赖的双链断裂(DSB)修复。由于DSB通路根据细胞周期的不同阶段而不同，在第一个SPP2127资助期，我们已经证明了DSB的精确修复也发生在有丝分裂后神经元中。此外，DSB途径的修饰进一步改善了精确修复。我们还确定了人类干细胞来源的神经元是一个足够的体外试验台，用于测试精确基因组编辑的所有实验参数。此外，在健康和患病的人类和小鼠光感受器中，DSB活性仍在发挥作用，没有改变，并显示出与人类神经元高活性的同源性。我们的数据表明，小鼠模型代表了用于治疗性基因编辑的复杂的体内模型。基于我们的发现，我们将组装所有用于DSB途径工程的分子工具、gRNAs、ABCA4模板和DSB报告结构，这些工具将被系统地应用于人类诱导的神经元，以揭示精确修复的最佳参数。我们还将产生一种ABCA4突变的人类干细胞系，我们将使用它来产生视网膜器官。这些3D人类视网膜有机体含有大量的光感受器，我们将通过AAVs来靶向这些感光器，以提供基因校正所需的所有组件。我们将结合成像、转录、基因组和定量蛋白质组读数来深入研究ABCA4修复。最终，我们还将在Stargardt病小鼠模型中测试我们的方法，以在体内纠正ABCA4基因。治疗后的小鼠将使用实时成像、行为测试和电生理学进行研究。下一代测序将揭示目标上和潜在的偏离目标的影响。在体内证明了有效性和安全性，并使用复杂的人类体外模型来纠正ABCA4基因座，简化了临床翻译并为其铺平了道路。我们对包括DSB途径工程在内的精确基因编辑的概念验证研究也将对IRD的其他治疗干预具有指导意义，而且一般也对有丝分裂后神经元的基因组工程具有指导意义。

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases (IRD). Gene-based therapies, mostly in the form of gene addition or supplementation therapies using adeno-associated viral (AAV) vectors for gene delivery, have been developed for several IRDs. However, all so far targeted diseases are associated with mutations in genes small enough that the corresponding cDNA can be transferred in a single AAV. Unfortunately, many of the most frequently mutated genes such as ABCA4 exceed the AAV cargo size. ABCA4 mutations cause Stargardt disease, an early onset form of macular degeneration. As gene therapies are hampered for Stargardt disease due to ABCA4 sequence length, gene editing represents an attractive approach to correct the mutation in the genomes of the patient’s photoreceptors. Precise gene editing, to avoid unwanted and uncontrolled additional genomic alterations, requires homology-dependent double strand break (DSB) repair. As DSB pathways differ according to the cell cycle’s stage, in the first SPP2127 funding period, we have demonstrated that precise DSB repair also occurs in postmitotic neurons. In addition, DSB pathway modifications further improved precise repair. We have also identified human stem cell-derived neurons as an adequate in vitro testbed for testing all experimental parameters for precise genome editing. In addition, the DSB activity in healthy and diseased human and mouse photoreceptors is at work, unaltered and shows a high activity homology to human neurons. Our data suggest that mouse models represent sophisticated in vivo models for therapeutic gene editing.Based on our findings, we will assemble all molecular tools for DSB pathway engineering, gRNAs, ABCA4 templates and DSB reporter constructs, which will be systematically applied to human induced neurons to reveal the optimal parameters for precise repair. We will also generate an ABCA4 mutated human stem cell line that we will use for generating retinal organoids. These 3D human retinal organoids contain lots of photoreceptors that we will target by AAVs to deliver all necessary components for gene correction. We will combine imaging, transcriptomic, genomic and quantitative proteomic readouts to study the ABCA4 repair in depth. Ultimately, we will test our approach also in a Stargardt disease mouse model to correct the Abca4 gene in vivo. Treated mice will be studied using live imaging, behavior testing and electrophysiology. On- and potential off-target effects will be revealed by next generation sequencing. Demonstrating in vivo efficacy and safety as well as employing sophisticated human in vitro models for correcting the ABCA4 locus streamlines and paves the way for clinical translation. Our proof-of-concept study for precise gene editing including DSB pathway engineering will also be instructive for other therapeutic interventions for IRDs but also in general for genomic engineering of postmitotic neurons.