Email Alert | RSS

Journal of Peking University (Health Sciences) ›› 2020, Vol. 52 ›› Issue (5): 845-850. doi: 10.19723/j.issn.1671-167X.2020.05.008

Previous Articles     Next Articles

Expression pattern of different serotypes of adeno-associated viral vectors in mouse retina

Shuang HU,Li-ping YANG()   

  1. Department of Ophthalmology, Peking University Third Hospital, Beijing 100191, China
  • Received:2018-04-13 Online:2020-10-18 Published:2020-10-15
  • Contact: Li-ping YANG E-mail:alexlipingyang@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(81470666);National Natural Science Foundation of China(81770966)

RICH HTML

   

PDF (PC)

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

CLC Number: 

  • R774.1

Figure 1

Subretinal injection schematis and subretinal injection under the microscope A, subretinal injection schematis, the mice lateral limbus was focused with a ligation tweezer, and the needle was injected from temporal side, the AAVs were injected into the contralateral retina; B, under the microscope, the treated part of retina turns to yellowish green and retinal blood vessels were visible, showing that drugs were injected into the subretinal space correctly. "

Figure 2

Expression of GFP reporter gene in different AAV serotype in mouse retina (×40) A, AAV2/2 showed weak GFP fluorescence (with DAPI marked nuclear); B, AAV2/2 showed weak GFP fluorescence; C, AAV2/5 showed little GFP fluorescence(with DAPI marked nuclear); D, AAV2/5 showed little GFP fluorescence; E, AAV2/8 showed specific GFP fluorescence for photorecepter (with DAPI marked nuclear); F, AAV2/8 showed specific GFP fluorescence for photoreceptor; G, AAV2/9 show GFP fluorescence expression in each layer of the retina including ganglion cell (with DAPI marked nuclear); H, AAV2/9 show GFP fluorescence expression in each layer of the retina including ganglion cell. GFP is green; DAPI is blue. AAV, adeno-associated virus; GFP, green fluorescent protein; DAPI, 4’, 6-diamidino-2-phenylindole; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. "

Figure 3

GFP reporter gene expression after 2 weeks and 4 weeks subretinal injection of AAV8-CMV (×40) A, AAV8 GFP fluorescence expression at 2 weeks (with DAPI marked nuclear); B, AAV8 GFP fluorescence expression at 2 weeks; C, AAV8 GFP fluorescence expression at 4 weeks showed parallel GFP fluorescence as 2 weeks (with DAPI marked nuclear); D, AAV8 GFP fluorescence expression at 4 weeks. GFP is green; DAPI is blue. W, week; AAV, adeno-associated virus; GFP, green fluorescent protein; DAPI, 4’, 6-diamidino-2-phenylindole. "

Figure 4

GFP reporter gene in different promoters (×20) A,AAV8 with CAG showed strong GFP fluorescence expression in photorecepter(with DAPI marked nuclear); B, AAV8 with CAG showed strong GFP fluorescence expression in photoreceptor; C, AAV8 with CMV showed strong GFP fluorescence expression both in photoreceptor and RPE(with DAPI marked nuclear); D, AAV8 with CMV showed strong GFP fluorescence expression both in photoreceptor and retinal pigment epithelium. GFP is green; DAPI is blue. AAV, adeno-associated virus; GFP, green fluorescent protein; DAPI, 4’, 6-diamidino-2-phenylindole. "

[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
[1] JIA Rui-xuan,JIANG Shang-wei,ZHAO Lin,YANG Li-ping. Generation and characterization of Cyp4v3 gene knockout mice [J]. Journal of Peking University (Health Sciences), 2021, 53(6): 1099-1106.
[2] Xiao-zhen LIU,Ying-ying LI,Li-ping YANG. Comparison study of whole exome sequencing and targeted panel sequencing in molecular diagnosis of inherited retinal dystrophies [J]. Journal of Peking University (Health Sciences), 2020, 52(5): 836-844.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Copyright © Journal of Peking University (Health Sciences), All Rights Reserved.
Powered by Beijing Magtech Co. Ltd