Boye Shannon E, Alexander John J, Witherspoon C Douglas, Boye Sanford L, Peterson James J, Clark Mark E, Sandefer Kristen J, Girkin Chris A, Hauswirth William W, Gamlin Paul D
1 Department of Ophthalmology, University of Florida College of Medicine , Gainesville, Florida.
2 Department of Human Genetics, Emory University , Atlanta, Georgia.
Hum Gene Ther. 2016 Aug;27(8):580-97. doi: 10.1089/hum.2016.085.
Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes are greatly enhanced.
腺相关病毒（AAV）已成为将基因表达靶向视网膜的首选载体。视网膜下注射的AAV可有效转导灵长类动物视网膜中的视网膜色素上皮和光感受器。玻璃体内递送的AAV可转导灵长类动物的内视网膜和中视网膜，但效率较低。这是由于载体稀释、衣壳可能被中和（因为它不限于免疫赦免的视网膜区域）以及存在将玻璃体与神经视网膜分隔开的内界膜（ILM）。我们在此描述一种解决所有这三个问题的新型“ILM下”注射方法。具体而言，将载体置于ILM与神经视网膜之间通过手术诱导的水分离空间中。在最初的实验中，我们注射了粘弹性物质（Healon®），我们证实该物质与AAV具有生物相容性，以形成ILM下泡，随后在用基本盐溶液冲洗后将AAV2-GFP注入泡中。对于后续实验，我们使用Healon-AAV混合物进行单次ILM下注射。在所有情况下，AAV的ILM下递送耐受性良好——通过眼科检查或光学相干断层扫描未观察到炎症或明显的结构变化。活体荧光成像显示在ILM下注射泡区域内有深刻的转基因表达，且在研究期间持续存在。在ILM下泡下方区域实现了视网膜神经节细胞（RGC）的均匀且广泛的转导。还观察到了米勒胶质细胞、ON双极细胞和光感受器的转导。来自表达绿色荧光蛋白的RGC的强大中枢标记证实了它们的持续存活，并在外侧膝状体、上丘和顶盖前区观察到。我们的结果证实，ILM是AAV在灵长类动物视网膜中转导的主要障碍，并且当绕过该障碍时，AAV2促进多种视网膜细胞类型转导的效率和深度会大大提高。
