A rAAV2/6 Mutant with Enhanced Targeting for Mouse Retinal Muller Cells
A rAAV2/6 Mutant with Enhanced Targeting for Mouse Retinal Muller Cells
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具有增强小鼠视网膜 Müller 细胞靶向性的 rAAV2/6 突变体
DOI：
10.1080/02713683.2019.1639768
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发表时间：
2019-08-25
影响因子：
2
通讯作者：
Zhang, Chun
中科院分区：
文献类型：
--
作者：
Cao, Jinjing;Liu, Xiaomei;Zhang, Chun
目的：腺相关病毒载体(AAV)是目前视网膜基因治疗中应用最广泛的基因载体。Muller细胞在维持动态平衡和神经元结构的完整性、稳定性方面发挥着重要作用，并已被发现与许多视网膜病变有关。本研究的目的是鉴定一个rAAV2/6突变体，该突变体增加了对小鼠视网膜Muller细胞的趋向性。材料和方法：采用氨基酸诱变技术，构建了rAAV2/6衣壳突变体rAAV2/6-S663L。采用体内成像法和视网膜平板贴片法分析rAAV2/6-S663L和wt rAAV2/6基因在小鼠视网膜组织中的表达。通过视网膜组织冰冻切片、免疫组织化学(IHC)、Muller细胞特异性启动子调控的基因表达和双AAV荧光蛋白共表达来确定rAAV2/6-S663L对小鼠视网膜Muller细胞的靶向性。结果：活体成像、视网膜平板贴壁和视网膜组织冰冻切片结果显示，rAAV2/6-S663L和wt rAAV2/6在小鼠视网膜中有不同的特异性取向，rAAV2/6-S663L更优先靶向Muller细胞。Muller细胞特异性启动子控制的基因表达实验和IHC实验证实rAAV2/6-S663L比rAAV2/6更容易感染Muller细胞。在GFAP启动子控制下表达EGFP的rAAV2/6-S663L和CMV启动子控制下表达mCherry的rAAV2/6-S663L共感染小鼠视网膜，发现两种荧光蛋白在Muller细胞中共表达。结论：rAAV2/6-S663L比rAAV2/6对Muller细胞具有更强的趋向性，为视网膜疾病的基因治疗提供了新的有用工具。
Purpose: Adeno-associated virus vector (AAV) is the most accepted gene delivery vector for retinal gene therapy. Muller cells play an important role in maintaining homeostasis and neuronal structural integrity, stability and it has been found to be involved in many retinopathies. The aim of this study is to identify a rAAV2/6 mutant which has increased tropism for Muller cell of the mouse retina. Materials and Methods: Using amino acid mutagenesis, we created a rAAV2/6 capsid mutant, rAAV2/6-S663L. In vivo imaging and retinal flat mount were employed to analyze the gene expression of rAAV2/6-S663L and wt rAAV2/6 in mouse retinal tissue. Retinal tissue cryosection, immunohistochemistry (IHC), Muller cell-specific promoter-controlled gene expression, and double AAV fluorescent protein co-expression were performed to determine the targeting of rAAV2/6-S663L for mouse retinal Muller cells. Results: In vivo imaging, retinal flat mount and retinal tissue cryosection results showed that rAAV2/6-S663L and wt rAAV2/6 have different specific tropisms in mouse retina and rAAV2/6-S663L is more preferentially targeting Muller cells. Muller cell-specific promoter-controlled gene expression experiments and IHC test confirmed that rAAV2/6-S663L has a higher tendency to infect Muller cells than wt rAAV2/6. Co-infection of the mouse retina with one rAAV2/6-S663L expressing EGFP under the control of GFAP promoter and the other one expressing mCherry under the control of CMV promoter revealed co-expression of the two fluorescent proteins in Muller cells. Conclusions: The results confirmed that rAAV2/6-S663L has a higher tropism for Muller cells than wt rAAV2/6. Our findings could add a new useful tool for retinal disease gene therapy.