June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
CRISPR/Cas9 mutagenesis of Tetraspanin-10, a novel AMD risk variant, impacts human retinal pigment epithelial cell pigmentation and oxidative stress
Author Affiliations & Notes
  • Rebecca Kaye
    Clinical & Experimental Sciences, Ophthalmology, University of Southampton, Southampton, Hampshire, United Kingdom
  • Jorn Lakowski
    Clinical & Experimental Sciences, Ophthalmology, University of Southampton, Southampton, Hampshire, United Kingdom
  • Andrew J Lotery
    Clinical & Experimental Sciences, Ophthalmology, University of Southampton, Southampton, Hampshire, United Kingdom
  • Footnotes
    Commercial Relationships   Rebecca Kaye None; Jorn Lakowski None; Andrew Lotery Novartis, Allergan, Apellis, Code C (Consultant/Contractor), Gyroscope Therapeutics, Code I (Personal Financial Interest)
  • Footnotes
    Support  Wellcome Trust Grant Ref. 210572/Z/18/Z
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3197. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Rebecca Kaye, Jorn Lakowski, Andrew J Lotery; CRISPR/Cas9 mutagenesis of Tetraspanin-10, a novel AMD risk variant, impacts human retinal pigment epithelial cell pigmentation and oxidative stress. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3197.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Genetic risk is a key component of Age-Related Macular Degeneration (AMD) susceptibility. A novel single nucleotide polymorphism at Tetraspanin 10 (TSPAN10) has been associated with increased AMD risk. In the retina TSPAN10 is localized to the retinal pigment epithelium (RPE). The role TSPAN10 has in the RPE, and how it increases AMD risk is currently unknown. For the first time, we investigated the role of TSPAN10 in the RPE using CRISPR/Cas9 mutagenesis.

Methods : CRISPR/Cas9-mediated genome-editing was used to generate TSPAN10 gene knockouts in the Mastershef Human Embryonic Stem Cell (hESC) Line. We differentiated RPE from two TSPAN10 knock-out hESC lines and a normal parental line, three differentiations were created for each clone, (N=9). RPE cells were characterised by morphology, pigmentation, trans-epithelial electrical resistance (TEER), RNA sequencing and analysis of proteins associated with differentiated RPE using immunostaining and western blotting. Statistical analysis for RNA-seq was performed with DESeq2R package, ELISAs with one-way ANOVA, and quantitative western blotting with student’s two tailed t-test.

Results : Transcriptomic analysis of control and TSPAN10-/- RPE showed pigmentation, oxidative stress and protein misfolding pathways to be affected in TSPAN10-/- RPE cells. TSPAN10-/- RPE were characterised by significantly reduced pigmentation and tyrosinase production, analysed using transmission electron microscopy to assess melanosome numbers (p <0.01) and quantitative western blotting (p <0.01). Furthermore, their oxidative stress response was reduced, specifically, the expression and production of the antioxidative enzyme catalase (padj= 1.39 x 10-20), confirmed using western blotting and ELISA (p<0.0001).

Conclusions : Our results reveal a role for TSPAN10 in the RPE and hint at a novel pathway in AMD development. TSPAN10 appears to be involved in RPE pigmentation, concurrent with its association with hair and eye colour. TSPAN10’s involvement in the oxidative stress response of the RPE highlights its possible role in AMD. Further work is needed to understand how TSPAN10 exerts such effects and if it can be targeted for therapeutic purposes.

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

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×