Zhang Hanmeng, Sajdak Benjamin S, Merriman Dana K, Carroll Joseph, Lipinski Daniel M
Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Department of Ophthalmology and Visual Sciences, Medical College of Wisconsin, 925 N 87 Street, Milwaukee, WI 53226, USA.
Mol Ther Methods Clin Dev. 2021 Jun 12;22:96-106. doi: 10.1016/j.omtm.2021.06.005. eCollection 2021 Sep 10.
Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a "pre-retinal" injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.
玻璃体内注射是眼部基因递送中应用最广泛的注射技术。然而，载体扩散会受到玻璃体中的物理屏障和中和抗体的影响。与人类一样，13条纹地松鼠（13-LGS）的玻璃体相对体积比大鼠和小鼠等更常见的啮齿动物模型更大，这会进一步降低玻璃体内注射途径的转导效率。我们在此报告一种“视网膜前”注射方法，该方法可导致后透明膜脱离，并将载体递送至玻璃体和视网膜内层之间的空间。将携带普遍表达的mCherry报告基因的载体注射到成年野生型13-LGS的深层玻璃体或视网膜前空间中。然后，通过非侵入性成像、免疫荧光和流式细胞术评估腺相关病毒（AAV）介导的mCherry表达。与深层玻璃体递送相比，视网膜前给药以较低的载体剂量体积实现了全视网膜基因表达，并显著增加了转导的视锥光感受器的数量。这些结果表明，视网膜前注射是动物模型基因治疗策略开发中的一种有前景的工具，也是一种潜在的用于人类研究的方法，特别是对于后透明膜完整且玻璃体稳定较年轻个体。
