Wu Wenjing, McRae Joel, Brown Asa, Agbaga Martin-Paul
Department of Ophthalmology, Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States.
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States.
Invest Ophthalmol Vis Sci. 2025 Sep 2;66(12):18. doi: 10.1167/iovs.66.12.18.
Adeno-associated viruses (AAVs) have become the preferred vector for gene therapy in ophthalmology. However, requirements for specific cell surface receptors limit AAV-mediated retinal cell transduction efficiency. This led to the need to engineer novel AAV vectors for widespread retinal transduction and transgene expression. However, no comparative analyses of these novel AAV serotypes have been reported. Here, we compare the retinal transduction efficiency of four novel AAV serotypes in wild-type mice retina after intravitreal and subretinal injections.
In total, 1.0 µL each of the four different AAV/cytomegalovirus/enhanced green fluorescent protein (EGFP) synthetic serotypes (1 × 109 genome copies [GC]/eye) was delivered by intravitreal or subretinal injection into mouse eyes. Tropism of each serotype to efficiently transduce photoreceptor (PR) and retinal pigment epithelial (RPE) cells was examined by EGFP expression using fundoscopy and immunolabeling 1 and 2 months after administration. Retinal function was evaluated using electroretinography and optomotor kinetics.
Fundoscopy and immunolabeling of EGFP in both subretinally and intravitreally injected AAV/DJ and AAV/DJ8 retinas showed the highest transduction efficiency. Compared to intravitreal delivery, all serotypes successfully transduced PR and RPE cells after subretinal injections. However, only intravitreally delivered AAV27m8, AAV/DJ, and AAV/DJ8 efficiently transduce PRs. AAV/DJ8 exhibited the highest PR and RPE transduction of the four serotypes. Visual function was unaffected, and adverse immunologic responses were not observed between the serotype and the PBS-injected eye.
Synthetic AAV serotypes differentially transduced PR and RPE cells depending on the delivery route. AAV/DJ8 exhibited the most efficient transduction of PR and RPE cells when injected intravitreally.
腺相关病毒（AAV）已成为眼科基因治疗的首选载体。然而，对特定细胞表面受体的需求限制了AAV介导的视网膜细胞转导效率。这导致需要构建新型AAV载体以实现广泛的视网膜转导和转基因表达。然而，尚未有对这些新型AAV血清型的比较分析报道。在此，我们比较了四种新型AAV血清型在玻璃体内和视网膜下注射后在野生型小鼠视网膜中的转导效率。
将四种不同的AAV/巨细胞病毒/增强型绿色荧光蛋白（EGFP）合成血清型（1×10⁹基因组拷贝数[GC]/眼）各1.0µL通过玻璃体内或视网膜下注射到小鼠眼中。给药后1个月和2个月，通过眼底镜检查和免疫标记，利用EGFP表达检测每种血清型对光感受器（PR）和视网膜色素上皮（RPE）细胞的转导嗜性。使用视网膜电图和视动动力学评估视网膜功能。
视网膜下和玻璃体内注射的AAV/DJ和AAV/DJ8视网膜中EGFP的眼底镜检查和免疫标记显示转导效率最高。与玻璃体内给药相比，所有血清型在视网膜下注射后均成功转导了PR和RPE细胞。然而，只有玻璃体内注射的AAV27m8、AAV/DJ和AAV/DJ8能有效转导PR。AAV/DJ8在四种血清型中对PR和RPE的转导效率最高。视觉功能未受影响，且在血清型与注射磷酸盐缓冲液的眼睛之间未观察到不良免疫反应。
合成AAV血清型根据给药途径不同地转导PR和RPE细胞。玻璃体内注射时，AAV/DJ8对PR和RPE细胞的转导效率最高。
