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

优化光感受器 ABCA4 位点同源依赖性治疗基因组编辑的途径工程

• 批准号: 399432863
• 负责人: Professor Dr. Volker Busskamp, Ph.D.
• 金额: --
• 依托单位: Universitäts-Augenklinik Bonn
• 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/399432863?language=en
• 关键词: Pathway; engineering; optimize; homology; dependent

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.

超过200种视网膜特异性基因的突变与遗传性视网膜疾病（IRD）有关。基于基因的治疗，主要是以基因添加或补充治疗的形式，使用腺相关病毒（AAV）载体进行基因递送，已开发用于几种IRD。然而，到目前为止，所有靶向疾病都与足够小的基因突变相关，使得相应的cDNA可以在单个AAV中转移。不幸的是，许多最频繁突变的基因如ABCA 4超过了AAV货物大小。ABCA4突变导致Stargardt病，一种早期发作的黄斑变性形式。由于ABCA4序列长度阻碍了Stargardt病的基因治疗，基因编辑代表了一种有吸引力的方法来纠正患者光感受器基因组中的突变。精确的基因编辑，以避免不必要的和不受控制的额外的基因组改变，需要同源依赖性双链断裂（DSB）修复。由于DSB途径根据细胞周期的阶段而不同，在第一个SPP 2127资助期，我们已经证明了精确的DSB修复也发生在有丝分裂后的神经元中。此外，DSB途径修饰进一步改善了精确修复。我们还确定了人类干细胞衍生的神经元作为测试精确基因组编辑的所有实验参数的适当体外测试平台。此外，在健康和患病的人类和小鼠光感受器中的DSB活性是在起作用的，未改变的，并且显示出与人类神经元的高活性同源性。我们的数据表明，小鼠模型代表了治疗性基因编辑的复杂体内模型。基于我们的研究结果，我们将组装DSB通路工程的所有分子工具，gRNA，ABCA 4模板和DSB报告基因构建体，将其系统地应用于人类诱导神经元，以揭示精确修复的最佳参数。我们还将产生一个ABCA4突变的人类干细胞系，我们将用于产生视网膜类器官。这些3D人类视网膜类器官含有大量的光感受器，我们将通过AAV靶向这些光感受器，以提供基因校正所需的所有成分。我们将结合联合收割机成像，转录组学，基因组学和定量蛋白质组学读数，深入研究ABCA 4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。将使用活体成像、行为测试和电生理学研究经处理的小鼠。下一代测序将揭示靶向效应和潜在的脱靶效应。证明体内功效和安全性以及采用复杂的人类体外模型来校正ABCA 4基因座简化并为临床转化铺平道路。我们对精确基因编辑（包括DSB通路工程）的概念验证研究也将对IRD的其他治疗干预措施具有指导意义，而且通常对有丝分裂后神经元的基因组工程也具有指导意义。