Völkner Manuela, Pavlou Marina, Büning Hildegard, Michalakis Stylianos, Karl Mike O
German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany.
Department of Ophthalmology, University Hospital, LMU Munich, Munich, Germany.
Hum Gene Ther. 2021 Jul;32(13-14):694-706. doi: 10.1089/hum.2020.321. Epub 2021 Jun 29.
The most widely used vectors for gene delivery in the retina are recombinant adeno-associated virus (rAAV) vectors. They have proven to be safe and effective in retinal gene therapy studies aimed to treat inherited retinal dystrophies, although with various limitations in transduction efficiency. Novel variants with modified capsid sequences have been engineered to improve transduction and overcome limitations of naturally occurring variants. Although preclinical evaluation of rAAV vectors based on such novel capsids is mostly done in animal models, the use of human induced pluripotent stem cell (hiPSC)-derived organoids offers an accessible and abundant human testing platform for rAAV evaluation. In this study, we tested the novel capsids, AAV9.GL and AAV9.NN, for their tropism and transduction efficiency in hiPSC-derived human retinal organoids (HROs) with all major neuronal and glial cell types in a laminated structure. These variants are based on the AAV9 capsid and were engineered to display specific surface-exposed peptide sequences, previously shown to improve the retinal transduction properties in the context of AAV2. To this end, HROs were transduced with increasing concentrations of rAAV9, rAAV9.GL, or rAAV9.NN carrying a self-complementary genome with a cytomegalovirus-enhanced green fluorescent protein (eGFP) cassette and were monitored for eGFP expression. The rAAV vectors transduced HROs in a dose-dependent manner, with rAAV9.NN achieving the highest efficiency and fastest onset kinetics, leading to detectable eGFP signals in photoreceptors, some interneurons, and Müller glia already at 2 days post-transduction. The potency-enhancing effect of the NN peptide insert was replicated when using the corresponding AAV2-based version (rAAV2.NN). Taken together, we report the application of an HRO system for screening novel AAV vectors and introduce novel vector candidates with enhanced transduction efficiency for human retinal cells.
视网膜基因递送中使用最广泛的载体是重组腺相关病毒（rAAV）载体。在旨在治疗遗传性视网膜营养不良的视网膜基因治疗研究中，它们已被证明是安全有效的，尽管在转导效率方面存在各种限制。已设计出具有修饰衣壳序列的新型变体，以提高转导效率并克服天然变体的局限性。尽管基于此类新型衣壳的rAAV载体的临床前评估大多在动物模型中进行，但使用人诱导多能干细胞（hiPSC）衍生的类器官为rAAV评估提供了一个可及且丰富的人体测试平台。在本研究中，我们测试了新型衣壳AAV9.GL和AAV9.NN在hiPSC衍生的人视网膜类器官（HRO）中的嗜性和转导效率，该类器官具有层状结构，包含所有主要的神经元和神经胶质细胞类型。这些变体基于AAV9衣壳，并经过工程改造以展示特定的表面暴露肽序列，先前已证明这些序列在AAV2的背景下可改善视网膜转导特性。为此，用携带巨细胞病毒增强型绿色荧光蛋白（eGFP）盒的自我互补基因组的rAAV9、rAAV9.GL或rAAV9.NN的递增浓度转导HRO，并监测eGFP表达。rAAV载体以剂量依赖方式转导HRO，rAAV9.NN实现了最高效率和最快的起效动力学，在转导后2天，光感受器、一些中间神经元和穆勒胶质细胞中就已检测到eGFP信号。当使用相应的基于AAV2的版本（rAAV2.NN）时，NN肽插入的效力增强作用得以重现。综上所述，我们报告了一种用于筛选新型AAV载体的HRO系统的应用，并引入了对人视网膜细胞转导效率增强的新型载体候选物。
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