June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Fast generation of a nonhuman-primate retinitis pigmentosa model by knocking down TMEM138 expression using CasRx system
Author Affiliations & Notes
  • xie LiJing
    Sun Yat-Sen University Zhongshan Ophthalmic Center State Key Laboratory of Ophthalmology, Guangzhou, Guangdong, China
  • Dianlei Guo
    Sun Yat-Sen University Zhongshan Ophthalmic Center State Key Laboratory of Ophthalmology, Guangzhou, Guangdong, China
  • Lei Shi
    Sun Yat-Sen University Zhongshan Ophthalmic Center State Key Laboratory of Ophthalmology, Guangzhou, Guangdong, China
  • Chunqiao Liu
    Sun Yat-Sen University Zhongshan Ophthalmic Center State Key Laboratory of Ophthalmology, Guangzhou, Guangdong, China
  • Footnotes
    Commercial Relationships   xie LiJing None; Dianlei Guo None; Lei Shi None; Chunqiao Liu None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4881. doi:
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      xie LiJing, Dianlei Guo, Lei Shi, Chunqiao Liu; Fast generation of a nonhuman-primate retinitis pigmentosa model by knocking down TMEM138 expression using CasRx system. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4881.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Nonhuman primate (NHP) models of retinitis pigmentosa (RP) are crucial for understanding human inherited retinal diseases and important for therapeutic practices. Here, we applied adeno-associated virus (AAV)-delivery system combined with CasRx technology to knock down TMEM138 gene expression in the rhesus macaque retina to generate a NHP RP model.

Methods : Three mouse and monkey TMEM138 sgRNAs were designed on the Cas13design platform and cloned into AAV-CAG-CasRx-U6 vector. Plasmids were transfected into 293T cells to verify knockdown efficiency with RT-PCR. The highest knock-down efficiency construct was packaged into AAV virus and concentrated to a final titer of 5×1012–10×1012 virus genome/ml. 1×108 and 3 × 1013 viral particles were injected into the subretinal space of adult mice and monkeys, respectively. Fundus photography (FP) and optical coherence tomography (OCT) were used to monitor the changes of retinal anatomy, and full-field electroretinogram (ffERG) were used to evaluate retinal functions. Retinal histology was examined by immunohistochemistry.

Results : CasRx system knocked down at least 80% TMEM138 expression in cultured cells. Both mice and monkeys with TMEM138-knockdown showed degenerated photoreceptor outer segment and rhodopsin accumulation in the inner segment. OCT and Fundus photography of the monkey retinas showed thinner outer nuclear layer and retinal pigmentation at 2 and 3 months after AAV injection, respectively. ffERG exhibited declines in scotopic and photopic light responses at 2 months after viral injection. Introduction of a TMEM138 cDNA bearing alternative coding for several amino acids of the wild type protein into the monkey retina avoided CasRx sgRNA targeting and rescued the RP phenotypes.

Conclusions : Our results demonstrate that CasRx combined with AAV delivery system is able to specifically knock down TMEM138 gene expression in rhesus macaque retina, thus, established a nonhuman primate RP model. This is a fast and efficient approach to create NHP RP models, which retinal phenotypes resembled the genetically-engineered animals. The model further implies that CasRx system can be applied to elimination of RP genes with dominant mutations, meanwhile, rescue retinal defects through introduction of normal genes, and thus useful for gene therapy studies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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