June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Towards retinal regeneration: Reprogramming retinal glial cells into photoreceptors using CRISPRa
Author Affiliations & Notes
  • Raymond Wong
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Tu Nguyen
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Lyujie Fang
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Sam Lukowski
    Institute of Molecular Bioscience, University of Queensland, Queensland, Australia
  • Camden Lo
    Private consultancy, Victoria, Australia
  • Sandy Hung
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Chi D Luu
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Mark C Gillies
    Save Sight Institute, New South Wales, Australia
  • Alex W Hewitt
    Centre for Eye Research Australia / University of Melbourne, Victoria, Australia
  • Footnotes
    Commercial Relationships   Raymond Wong, None; Tu Nguyen, None; Lyujie Fang, None; Sam Lukowski, None; Camden Lo, None; Sandy Hung, None; Chi Luu, None; Mark Gillies, None; Alex Hewitt, None
  • Footnotes
    Support  NHMRC; Retina Australia; Centre for Eye Research Australia; University of Melbourne
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2497. doi:
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      Raymond Wong, Tu Nguyen, Lyujie Fang, Sam Lukowski, Camden Lo, Sandy Hung, Chi D Luu, Mark C Gillies, Alex W Hewitt; Towards retinal regeneration: Reprogramming retinal glial cells into photoreceptors using CRISPRa. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2497.

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

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Abstract

Purpose : The loss of photoreceptors is a key hallmark of many incurable blinding diseases. Previous studies showed the feasibility of using transcription factors to reprogram Müller glial cells to regenerate retinal neurons in mouse in vivo, providing an innovative approach to stimulate retinal regeneration. However, it remained unclear whether this reprogramming approach can translate to human cells. This study aims to use CRISPR activation (CRISPRa) to reprogram human Müller glial cells into photoreceptors (termed induced photoreceptors, iPH) in vitro.

Methods : We have optimized a CRISPRa system which allow us to activate efficient expression of 9 genes simultaneously. This platform greatly enhanced our capacity to perform in vitro screening in human Müller glial cell line (MIO-M1) to identify the optimal transcription factor cocktails that promote reprogramming into iPH. Validation of the iPH quality is performed by photoreceptor marker analysis using qPCR and immunocytochemistry, as well as single cell transcriptomic using 10x Chromium.

Results : As part of the Human Cell Atlas initiative (humancellatlas.org), we recently generated a human retina transcriptome atlas at single cell levels and identified the transcriptome of the major retinal cell types. From this dataset, we performed network topology analysis to predict and shortlisted 9 transcription factors that could reprogram Müller glial cells into photoreceptors. Using a CRISPRa platform, we have screened and identified cocktails of transcription factors that allow reprogramming of human Müller glial cells into iPH in vitro. qPCR and immunocytochemical analysis demonstrated that iPH expressed a panel of photoreceptor markers, including RHO and PDE6B. We performed single cell transcriptomic to profile iPH and compared with the human retina transcriptome atlas as a benchmark. Our results showed that iPH reprogramming promoted transcriptomic transitions from Müller glial cells to photoreceptors and activation of photoreceptor markers in iPH.

Conclusions : Our study demonstrated the use of CRISPRa to activate transcription factors and promote in vitro reprogramming of Müller glial cells into photoreceptors, which have implications in tissue engineering and regenerative medicine. Future application for in vivo reprogramming provides an exciting innovative approach to regenerate photoreceptors and restore vision in retinal degenerative diseases.

This is a 2020 ARVO Annual Meeting abstract.

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