不同血清型腺相关病毒载体转染小鼠视网膜后的表达效率

doi:10.19723/j.issn.1671-167X.2020.05.008

摘要/Abstract

摘要：

目的:研究不同血清型腺相关病毒 (adeno-associated virus,AAV) 载体介导的外源基因在视网膜中的表达效率, 同时比较AAV载体和两种眼科常用启动子组合后转染小鼠视网膜的表达效率高低,为视网膜色素变性基因治疗选择合适的AAV载体与启动子提供依据。方法:AAV病毒根据衣壳蛋白不同可分为不同血清型,本课题选取视网膜疾病基因治疗中常用的AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体,并以绿色荧光蛋白 (green fluorescent protein, GFP) 作为报告基因,用GFP的表达强度判断AAV载体介导的外源基因在视网膜中的表达效率。AAV载体纯化后滴度为1.00×10 ¹³ mg/L,注射1 μL至C57BL/6J小鼠视网膜下腔, 于2周取眼球做成冰冻切片,在共聚焦显微镜下观察 GFP在小鼠视网膜各层的表达情况。选取在感光细胞内特异性表达最强的AAV2/8于第4周取眼球冰冻切片继续观察是否能持续稳定表达。随后选取眼科基因治疗最常用的广谱启动子CMV和由CMV增强子与鸡β-肌动蛋白启动子组成的CAG启动子,并构建AAV2/8-GFP-CMV和AAV2/8-GFP-CAG两种不同启动子的病毒载体注射至视网膜下腔,于2周取眼球做成冰冻切片,在共聚焦显微镜下观察不同启动子的AAV2/8在小鼠视网膜各层的表达情况。结果: 注射AAV-GFP后未见典型的术后细菌感染及明显免疫反应。AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体视网膜下腔注射2周后,AAV2/8和AAV2/9在小鼠视网膜的GFP绿色荧光明显,说明这两种AAV载体转染小鼠视网膜后的表达效率高,而在这两种血清型中,AAV2/8的GFP绿色荧光主要集中在感光细胞内,AAV2/9 在视网膜全层均有表达,说明AAV2/8对视网膜感光细胞特异性更强。对AAV2/8的进一步实验表明在视网膜下腔注射4周后小鼠视网膜的GFP绿色荧光明显,说明AAV2/8载体介导的外源基因能在体内稳定表达。使用CMV启动子时GFP在感光细胞与视网膜色素上皮细胞均有表达,而使用CAG启动子时GFP主要在感光细胞表达。结论:视网膜下腔注射AAV病毒载体可在视网膜细胞内稳定表达报告基因;AAV2/2、AAV2/5、AAV2/8和AAV2/9四种血清型AAV载体中,AAV2/8和AAV2/9在视网膜表达能力最强,AAV2/8对视网膜感光细胞特异性最好;CMV和CAG两种启动子,CAG启动子对感光细胞特异性更高。

关键词: 腺相关病毒, 视网膜色素变性, 基因治疗

Abstract:

Objective: To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa. Methods: AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×10 ¹³ mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope. Results: No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells,while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells. Conclusion: AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.

Key words: Serotype adeno-associated viral, Retinitis pigmentosa, Gene therapy

中图分类号:

R774.1

胡双,杨丽萍. 不同血清型腺相关病毒载体转染小鼠视网膜后的表达效率[J]. 北京大学学报（医学版）, 2020, 52(5): 845-850.

Shuang HU,Li-ping YANG. Expression pattern of different serotypes of adeno-associated viral vectors in mouse retina[J]. Journal of Peking University (Health Sciences), 2020, 52(5): 845-850.

图/表 4

图1

图2

图3

图4

参考文献 19

[1]	Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors [J]. Nat Protoc, 2006,1(3):1412-1428. doi: 10.1038/nprot.2006.207 pmid: 17406430
[2]	Berns KI, Nicholas M. AAV: An overview of unanswered questions[J]. Hum Gene Ther, 2017,28(4):308-313. doi: 10.1089/hum.2017.048 pmid: 28335618
[3]	Alves CH, Wijnholds J. AAV gene augmentation therapy for CRB1-associated retinitis pigmentosa[J]. Methods Mol Biol, 2018,1715:135-151. pmid: 29188511
[4]	Moore NA, Morral N, Ciulla TA. Gene therapy for inherited retinal and optic nerve degenerations[J]. Expert Opin Biol Ther, 2018,18(1):37-49. doi: 10.1080/14712598.2018.1389886 pmid: 29057663
[5]	Sullivan JA, Stanek LM, Lukason MJ. Rationally designed AAV2 and AAVrh8R capsids provide improved transduction in the retina and brain[J]. Gene Ther, 2018,25(3):205-219. doi: 10.1038/s41434-018-0017-8 pmid: 29785047
[6]	Ong T, Pennesi ME, Birch DG. Adeno-Associated viral gene therapy for inherited retinal disease[J]. Pharm Res, 2019,36(2):34. doi: 10.1007/s11095-018-2564-5 pmid: 30617669
[7]	Russell S, Bennett J, Wellman JA. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: A randomised, controlled, open-label, phase 3 trial[J]. Lancet, 2017,390(10097):849-860. doi: 10.1016/S0140-6736(17)31868-8 pmid: 28712537
[8]	Hung SC, Chrysostomou V, Li F. AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo[J]. Invest Ophthalmol Vis Sci, 2016,57(7):3470-3476. doi: 10.1167/iovs.16-19316 pmid: 27367513
[9]	Day TP, Byrne LC, Schaffer DV, et al. Advances in AAV vector development for gene therapy in the retina[J]. Adv Exp Med Biol, 2014,801:687-693. doi: 10.1007/978-1-4614-3209-8_86
[10]	Allocca M, Mussolino C, Garcia-Hoyos M, et al. Novel Adeno-associated virus serotypes efficiently transduce murine photoreceptors[J]. J Virol, 2007,81(20):11372-11380. doi: 10.1128/JVI.01327-07 pmid: 17699581
[11]	Surace EM, Auricchio A. Versatility of AAV vectors for retinal gene transfer[J]. Vision Res, 2008,48(3):353-359. doi: 10.1016/j.visres.2007.07.027
[12]	Bennett J, Wellman J, Marshall KA. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial[J]. Lancet, 2016,388(10045):661-672. doi: 10.1016/S0140-6736(16)30371-3 pmid: 27375040
[13]	Carvalho LS, Xu J, Pearson RA, et al. Long-term and age-dependent restoration of visual function in a mouse model of CNGB3-associated achromatopsia following gene therapy[J]. Hum Mol Genet, 2011,20(16):3161-3175. doi: 10.1093/hmg/ddr218
[14]	Flannery JG, Zolotukhin S, Vaquero MI. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus[J]. Proc Natl Acad Sci USA, 1997,94(13):6916-6921. doi: 10.1073/pnas.94.13.6916 pmid: 9192666
[15]	Young JE, Vogt T, Gross KW, et al. A short, highly active photoreceptor-specific enhancer/promoter region upstream of the human rhodopsin kinase gene[J]. Invest Ophthalmol Vis Sci, 2003,44(9):4076-4085. doi: 10.1167/iovs.03-0197 pmid: 12939331
[16]	Nicoletti A, Kawase K, Thompson DA. Promoter analysis of RPE65, the gene encoding a 61-kDa retinal pigment epithelium-specific protein[J]. Invest Ophthalmol Vis Sci, 1998,39(3):637-644. pmid: 9501877
[17]	Esumi N, Oshima Y, Li Y, et al. Analysis of the VMD2 promoter and implication of E-box binding factors in its regulation[J]. J Biol Chem, 2004,279(18):19064-19073. doi: 10.1074/jbc.M309881200 pmid: 14982938
[18]	Corti M, Liberati C, Smith BK. Safety of intradiaphragmatic deli-very of adeno-associated virus-mediated alpha-glucosidase (rAAV1-CMV-hGAA) gene therapy in children affected by pompe disease[J]. Hum Gene Ther Clin Dev, 2017,28(4):208-218. doi: 10.1089/humc.2017.146 pmid: 29160099
[19]	Martier R, Sogorb-Gonzalez M, Stricker-Shaver J. Development of an AAV-based MicroRNA gene therapy to treat Machado-Joseph disease[J]. Mol Ther Methods Clin Dev, 2019,15:343-358. doi: 10.1016/j.omtm.2019.10.008 pmid: 31828177

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed