June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Characterizing AMD risk genes in an iRPE functional genetic screening platform
Author Affiliations & Notes
  • Heshan Peiris
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Maggie Crow
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Frank Yang
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Celine Eidenschenk
    Discovery Oncology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Amy Stockwell
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Xiaoyu Hu
    Discovery Oncology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Jennifer Gilda
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
    Discovery Oncology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Zora Modrusan
    Microchemistry, Proteomics, Lipidomics and Next Generation Sequencing, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Russell Xie
    Discovery Oncology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Scott Martin
    Discovery Oncology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Mark McCarthy
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Luz Orozco
    Bioinformatics and Computational Biology, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Hsu-Hsin Chen
    Biomarker Discovery OMNI, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Brian Yaspan
    Human Genetics, Genentech USA Inc South San Francisco, South San Francisco, California, United States
  • Footnotes
    Commercial Relationships   Heshan Peiris Genentech, Code E (Employment); Maggie Crow Genentech, Code E (Employment); Frank Yang Genentech, Code E (Employment); Celine Eidenschenk Genentech, Code E (Employment); Amy Stockwell Genentech, Code E (Employment); Xiaoyu Hu Genentech, Code E (Employment); Jennifer Gilda Genentech, Code E (Employment); Zora Modrusan Genentech, Code E (Employment); Russell Xie Genentech, Code E (Employment); Scott Martin Genentech, Code E (Employment); Mark McCarthy Genentech, Code E (Employment); Luz Orozco Genentech, Code E (Employment); Hsu-Hsin Chen Genentech, Code E (Employment); Brian Yaspan Genentech, Code E (Employment)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2796. doi:
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      Heshan Peiris, Maggie Crow, Frank Yang, Celine Eidenschenk, Amy Stockwell, Xiaoyu Hu, Jennifer Gilda, Zora Modrusan, Russell Xie, Scott Martin, Mark McCarthy, Luz Orozco, Hsu-Hsin Chen, Brian Yaspan; Characterizing AMD risk genes in an iRPE functional genetic screening platform. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2796.

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

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Abstract

Purpose :
Age-related macular degeneration (AMD) is the third leading cause of vision loss and is
characterized by lipid-rich extracellular deposits and localized inflammation in the macula. Retinal Pigment Epithelial (RPE) cells progressively lose their ability to phagocytose photoreceptor outer segments, maintain tight-barrier function and become inflamed resulting in changes in morphology and function during the intermediate and later stages of AMD. Genome-wide association studies (GWAS) and transcriptomic profiling of human donor eyes strongly implicate RPEs in AMD risk, progression and pathogenesis. However, little is known about the gene programs driving AMD risk in the context of RPE biology.

Methods :
We have developed a functional genetic screening platform using human iPSC derived RPEs
(iRPEs). These cells recapitulate key RPE transcriptomic profiles when assessed via scRNA- and scATAC-seq. iRPEs also display functional aspects of RPEs such as pigmentation, phagocytosis, barrier function and secretion of immunomodulatory cytokines. To knock out any gene of interest we optimized a CRISPR-Cas9 mediated protocol which efficiently perturbed gene expression without compromising cell survival. We have thus developed an iRPE functional genetic screening platform which allows for scalable assessment of gene function (Figure 1).

Results :
To understand the gene programs driving AMD risk and progression we screened genes with population or functional genetic evidence of association with advanced or intermediate AMD, as well as the RPE transcription factor, MITF, as a positive control. MITF knockout resulted in a discernible change in the transcriptomic profile and dysregulation of pathways associated with melanogenesis, gap junction formation and MAPK signaling. Knockout of Nemo-like kinase (NLK) resulted in a significant reduction in the phagocytic activity of iRPE cells independent of changes in morphology or viability.

Conclusions : Together these data highlight the power of using functional genetic screens informed by human genetics in appropriate in vitro systems to uncover novel biology and for therapeutic target discovery. The intersection of human genetics and functional genetic screens provides a new paradigm for target prioritization and drug discovery.

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

 

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