June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
TYROSINASE Deficient Human Retinal Pigment Epithelium Exhibits Melanosome Maturation Defects
Author Affiliations & Notes
  • Arnold Leigh
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Aman George
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Tyler Pfister
    National Institutes of Health, Bethesda, Maryland, United States
  • Charles DeYoung
    National Institutes of Health, Bethesda, Maryland, United States
  • Ruchi Sharma
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Mones Abu-Asab
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Jizhong Zou
    National Institutes of Health, Bethesda, Maryland, United States
  • Kapil Bharti
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Brian Patrick Brooks
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Arnold Leigh None; Aman George None; Tyler Pfister None; Charles DeYoung None; Ruchi Sharma None; Mones Abu-Asab None; Jizhong Zou None; Kapil Bharti None; Brian Brooks None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 775. doi:
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      Arnold Leigh, Aman George, Tyler Pfister, Charles DeYoung, Ruchi Sharma, Mones Abu-Asab, Jizhong Zou, Kapil Bharti, Brian Patrick Brooks; TYROSINASE Deficient Human Retinal Pigment Epithelium Exhibits Melanosome Maturation Defects. Invest. Ophthalmol. Vis. Sci. 2023;64(8):775.

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

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Abstract

Purpose : Oculocutaneous albinism type 1A (OCA1A) is a rare recessive genetic condition due to mutations in TYROSINASE that result in pigmentation defects of the skin, hair, and eyes. The reduced or absent ocular pigmentation is associated with defects in fovea development resulting in reduced best-corrected visual acuity. In this study, we used CRISPR-Cas9 to knockout the TYROSINASE gene in induced pluripotent stem cells to generate an in vitro model for OCA1A.

Methods : All human iPSC work was approved by the NIH Institutional Review Board, protocol #11-E1-0245 (NCT01432847). Control iPSC line was reprogrammed using Sendai virus-mediated delivery (CytoTune2) of the four Yamanaka factors. iPSCs were cultured in Essential 8™ medium for expansion until the start of differentiation. iPSC lines were characterized for pluripotency by immuno-fluorescence staining using antibodies for OCT4, SOX2, NANOG, SSEA-4, TRA-1-81, and TRA-1-60. A 20nt guide RNA (gRNA) sequence targeting Exon1 of TYR was identified using an in-silico program and cloned into pCAG-eCas9-GFP-U6-gRNA plasmid vector (Addgene) to express gRNA, a high-fidelity version of SpCas9 (eCas9), and a green fluorescent protein (GFP) marker. 48 hours after transfection, GFP positive iPSCs were sorted onto Matrigel-coated 96-well plates containing 100 µL E8 and 1x CloneR supplement (STEMCELL Technologies) at one cell per well. Wells with surviving clones were expanded to isolate genomic DNA for screening. A genomic fragment spanning the gRNA target sites was PCR amplified and Sanger sequenced to identify TYR bi-allelic knockout clones. The TIDE online program was used to analyze Sanger sequencing results for compound heterozygous knockout clones.

Results : The TYROSINASE knock-out iPSC displayed normal human karyotype and expression of the pluripotency markers including NANOG, OCT4, SOX-2, SSEA4, TRA-1-60, and TRA-1-81. TYR-/--iRPE exhibited a complete lack of pigmentation and mature melanosomes, normal melanosome biogenesis, and abnormal apical junction organization. Trans-epithelial resistance and apical-basal polarity were comparable to isogenic pigmented RPE

Conclusions : TYROSINASE knockout iPSC forms a morphologically normal iRPE monolayer with proper apical-basal polarization and exhibits pigmentation defects similar to OCA1A patient-derived iRPE.

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

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