Investigative Ophthalmology & Visual Science Cover Image for Volume 65, Issue 7
June 2024
Volume 65, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2024
Modeling of GRM6-Associated Congenital Stationary Night Blindness in iPSC-Derived Human Retinal Organoids
Leila Bahmani; Jinlun Bai; Sumitha Prameela Bharathan; Rosanna Calderon; Esteban Fernandez; Kate Matsunaga; Kayla Stepanian; Patricia Galvan; Aaron Nagiel
Author Affiliations & Notes
  • Leila Bahmani
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Jinlun Bai
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    Department of Development, Stem Cells and Regenerative Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Sumitha Prameela Bharathan
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Rosanna Calderon
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    Department of Development, Stem Cells and Regenerative Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Esteban Fernandez
    The Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Kate Matsunaga
    University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Kayla Stepanian
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Patricia Galvan
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
  • Aaron Nagiel
    The Vision Center, Department of Surgery, Children's Hospital Los Angeles, Los Angeles, California, United States
    Roski Eye Institute, Department of Ophthalmology, University of Southern California Keck School of Medicine, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Leila Bahmani None; Jinlun Bai None; Sumitha Prameela Bharathan None; Rosanna Calderon None; Esteban Fernandez None; Kate Matsunaga None; Kayla Stepanian None; Patricia Galvan None; Aaron Nagiel None
  • Footnotes
    Support  an unrestricted grant to the Department of Ophthalmology at the Keck School of Medicine of USC from Research to Prevent Blindness, New York, NY, USA ,a National Eye Institute Career Development Award K08EY030924, the Las Madrinas Endowment in Experimental Therapeutics for Ophthalmology , a Research to Prevent Blindness Career Development Award , The Saban Research Institute of Children’s Hospital Los Angeles Pre-Doctoral Award, CIRM Training Program for Stem Cell and Regenerative Medicine Research
Investigative Ophthalmology & Visual Science June 2024, Vol.65, 1561. doi:
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      Leila Bahmani, Jinlun Bai, Sumitha Prameela Bharathan, Rosanna Calderon, Esteban Fernandez, Kate Matsunaga, Kayla Stepanian, Patricia Galvan, Aaron Nagiel; Modeling of GRM6-Associated Congenital Stationary Night Blindness in iPSC-Derived Human Retinal Organoids. Invest. Ophthalmol. Vis. Sci. 2024;65(7):1561.

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

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Abstract

Purpose : Congenital stationary night blindness (CSNB) is an incurable inherited retinal disease characterized by impaired signal transmission from the photoreceptors to bipolar cells. Mutations in the GRM6 gene, encoding metabotropic glutamate receptor 6 (mGluR6), are the third most prevalent cause of the complete form of CSNB. Despite the existence of animal models, there is still a need for human in vitro systems to model GRM6-associated CSNB and test potential therapies.

Methods : We analyzed previously published single-cell RNA sequencing (scRNAseq) datasets and then performed confirmatory RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence staining (IF) in the mouse retina, human fetal retina, and human retinal organoid (HRO) sections. A tamoxifen-inducible GRM6 knockout induced pluripotent stem cell (iPSC) line was created by combining CRISPR/Cas9-mediated gene editing with the Cre/LoxP system. HROs are being generated from these inducible knockout iPSCs to serve as a human GRM6-associated CSNB model.

Results : scRNAseq analysis showed GRM6 expression by bipolar cells in mouse and human retina. Interestingly, our scRNAseq analysis over multiple time points of mouse and human retinal development revealed expression of GRM6 by cone photoreceptors in the human fetal retina, unlike mouse retina where GRM6 expression was restricted to bipolar cells. GRM6 expression in the photoreceptor layer of human retina was confirmed using RNA-FISH and IF staining performed during synapse development on mouse retina (P14), human fetal retina (Hgw16), and HROs (Day 190). Sanger sequencing verified the biallelic knock-in of LoxP sequences flanking GRM6 exon 1 and 2, and ERT2CreERT2 in the AAVS1 locus (Figure 1). HROs generated from inducible knockout iPSCs line will permit precise temporal deletion of GRM6 upon tamoxifen treatment at any stage of HRO maturation.

Conclusions : This study analyzed GRM6 expression in developing mouse and human retina. Modeling of GRM6 deletion in human retina during synapse assembly and maintenance will be possible by utilizing tamoxifen-inducible GRM6 knockout HROs.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Generation of tamoxifen-inducible GRM6 knockout iPSCs.
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Generation of tamoxifen-inducible GRM6 knockout iPSCs.

Generation of tamoxifen-inducible GRM6 knockout iPSCs.

Generation of tamoxifen-inducible GRM6 knockout iPSCs.
View OriginalDownload Slide

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Copyright © 2025 Association for Research in Vision and Ophthalmology.
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