中文摘要

目的

构建靶向USH2A外显子13的反义寡核苷酸（AON）；开发并优化AON递送工具以改善AON体内递送效率。通过视网膜母细胞瘤（Rb1）细胞系筛选、人源化USH2A-e13转基因小鼠模型以及患者来源的视网膜类器官模型评估AON引起USH2A外显子13跳跃的效率，分析其治疗机制。同时探索USH2A基因变异的致病机制及其对感光细胞纤毛结构的影响。

方法

通过生物信息学设计与筛选靶向USH2A外显子13的AON，利用Rb1细胞在RNA和蛋白层面分析其跳跃效率。

利用基因编辑构建人源化USH2A-e13转基因小鼠模型，进行玻璃体腔注射AON，利用眼底荧光观察、组织切片荧光检测、ddPCR、Western Blot（WB）、凋亡检测和ERG评估其在视网膜组织中的分布、跳跃效率及毒性。

使用患者来源的iPSCs诱导分化形成视网膜类器官进行转录组分析，通过镜下观察、HE染色、电镜、免疫荧光染色探究USH2A相关遗传性视网膜变性视网膜类器官的病理改变。根据切片荧光检测、ddPCR、WB、免疫荧光染色和凋亡检测评估AON在感光细胞层中的分布、跳跃效率及毒性。通过验证转录组差异表达通路关键基因进一步研究AON对感光细胞中纤毛和微管结与功能的修复潜力，验证其在体外模型中的治疗机制。

开发并优化RNA纳米颗粒（RNP）和外泌体（EV）递送工具，利用荧光拍照、眼底荧光定位、切片荧光检测不同工具在Rb1细胞、野生型小鼠和类器官中的递送效率。

结果

1.       物理结构和化学修饰优化后的新型AON (PUMCH-E13) 在Rb1细胞系中实现了有效的USH2A外显子13跳跃。ddPCR结果显示，PUMCH-E13在高浓度（40 µM）下显示跳跃效率为 15.8 copies/µL，高于同浓度下的阳性对照QR421a跳跃效率（14.9 copies/µL）。

2.       USH2A-e13小鼠模型中，PUMCH-E13通过玻璃体腔注射实现了外显子13的跳跃，ddPCR显示阳性跳跃事件数量可达68.3%（40ug组），WB显示usherin蛋白的相对表达量较对照组降低44.44% ± 1.61%，并且凋亡检测和ERG未显示明显的不良影响。

3.       患者来源的视网膜类器官ddPCR数据显示，PUMCH-E13诱导外显子13跳跃效率可达6.49 copies/µL，WB发现PUMCH-E13治疗组中usherin蛋白外显子13区域的表达降低了16.4 ± 4.1%。PUMCH-E13治疗后视网膜感光细胞USH2复合体中GPR98和PDZD7在RNA和蛋白层面均表达回升，感光细胞纤毛中微管结构有所改善。

4.       两种递送工具（RNP和EV）均显示出较高的AON递送效率，其中EV-AON在Rb1细胞核内定位强，在小鼠视锥视杆细胞层表达更强烈，在患者视网膜类器官感光细胞层的荧光表达优于RNP-AON。

结论

1.       构建的新型AON (PUMCH-E13) 能够有效跳跃USH2A外显子13，并在Rb1细胞系、USH2A-e13小鼠模型和患者视网膜类器官中表现出显著的挽救效果和低毒性。

2.       患者来源视网膜类器官的研究表明PUMCH-E13在体外模型中能够促进视网膜感光细胞纤毛结构的恢复，揭示了其潜在的治疗机制。

3.       RNP和EV作为有效的递送工具能够提高AON的细胞内转染，其中EV-AON在感光细胞中的表现尤为突出。

Abstract

Purpose

To construct AON targeting USH2A exon 13; to develop and optimize AON delivery tools to improve AON in vivo transfection efficiency. To assess the efficiency of AON-induced USH2A exon 13 skipping and analyze the therapeutic mechanism through Rb1 cell, humanized USH2A-e13 transgenic mouse, and patient-derived retinal organoid model. We also explored the pathogenic mechanism of the USH2A gene variant and its effect on the ciliary structure of photoreceptor cells.

Methods

We designed and screened AON targeting USH2A exon 13 by bioinformatics, and analyzed its skipping efficiency at RNA and protein levels using Rb1 cells.

A humanized USH2A-e13 transgenic mouse model was constructed to assess the distribution , skipping efficiency and toxicity in retinal tissues after intravitreal injection of AON, by fundus fluorescence observation, fluorescence detection in tissue sections, ddPCR, WB and apoptosis assay.

Patient-derived iPSCs were induced and differentiated to retinal organoids. for transcriptome analysis and explored the effects of the USH2A gene variant on photoreceptor cells in retinal organoids by microscopic observation, HE staining, electron microscopy, and immunofluorescence staining. The distribution, skipping efficiency and toxicity of AON in the photoreceptor cell layer were assessed based on section fluorescence detection, ddPCR, WB, immunofluorescence staining and apoptosis assay. The repair potential of AON on cilia and microtubule junctions and functions in photoreceptor cells was further investigated by validating the key genes of the transcriptome differential expression pathway, and its therapeutic mechanism was verified in an in vitro model.

Develop and optimize RNA nanoparticle (RNP) and exosome (EV) delivery tools. Use fluorescence photography, fundus fluorescence localization, and section fluorescence to assess the delivery efficiency of different tools in Rb1 cells, wild-type mice, and organoids.

Results

The novel AON (PUMCH-E13) optimized for physical structure and chemical modification achieved efficient USH2A exon 13 jumping in Rb1 cell line. ddPCR results indicated that PUMCH-E13 displayed a jumping efficiency of 15.8 copies/µL at high concentration (40 µM group), which was higher than that of the positive control QR421a jumping efficiency (14.9 copies/µL) at the same concentration. (14.9 copies/µL).

In the USH2A-e13 mouse model, PUMCH-E13 was injected intravitreally to achieve exon 13 jumping. ddPCR revealed that the number of positive jumping events could reach 68.3% (40ug group), and WB demonstrated that the relative expression of usherin protein was reduced by 44.44% ± 1.61% compared with the control group, and apoptosis assay did not show any obvious toxic response. .

ddPCR data from patient-derived retinal organoids demonstrated that PUMCH-E13 induced exon 13 jumping efficiency up to 6.49 copies/µL, and WB revealed that the expression of the exon 13 region of the usherin protein was reduced by 16.4 ± 4.1% in the PUMCH-E13 group. Retinal photoreceptor cells after PUMCH-E13 treatment Expression of GPR98 and PDZD7 in the USH2 complex rebounded at both the RNA and protein levels, and microtubule structure in photoreceptor cell cilia was improved.

Both delivery tools (RNP and EV) demonstrated high AON delivery efficiency, with EV-AON being strongly localized in the nucleus of Rb1 cells, more strongly expressed in the mouse optic cone optic rod cell layer, and fluorescently expressed in the photoreceptor cell layer of the patient's retinal analogs better than RNP-AON.

Conclusions

The constructed novel AON (PUMCH-E13) was able to effectively jump USH2A exon 13 and indicated significant rescue，efficacy and low toxicity in the Rb1 cell line, the humanized USH2A-e13 transgenic mouse model, and patients’ retinal organoid.

Patient-derived retinal organoid studies demonstrated that PUMCH-E13 was able to promote the restoration of retinal photoreceptor cell cilia structure in an in vitro model, revealing its potential therapeutic mechanism.

RNP and EV, as effective delivery tools, were able to enhance the intracellular transfection of AON, with EV-AON being particularly prominent in photoreceptor cells.