June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Development of a Gene Augmentation Strategy for CRB1-mediated Inherited Retinal Diseases
Author Affiliations & Notes
  • Joel Alan Imventarza
    Ophthalmology, Columbia University, New York, New York, United States
  • Keith Theodore
    Ophthalmology, Columbia University, New York, New York, United States
  • Bruna Lopes da Costa
    Ophthalmology, Columbia University, New York, New York, United States
    Department of Biomedical Engineering, Columbia University, New York, New York, United States
  • Salvatore Marco Caruso
    Ophthalmology, Columbia University, New York, New York, United States
    Department of Biomedical Engineering, Columbia University, New York, New York, United States
  • Stephen H. Tsang
    Ophthalmology, Columbia University, New York, New York, United States
  • Yi-Ting Tsai
    Rejuvitas, New York, New York, United States
  • Peter M.J. Quinn
    Ophthalmology, Columbia University, New York, New York, United States
  • Footnotes
    Commercial Relationships   Joel Imventarza None; Keith Theodore None; Bruna Lopes da Costa None; Salvatore Caruso None; Stephen Tsang Columbia University, Code P (Patent); Yi-Ting Tsai Columbia University, Code P (Patent); Peter Quinn Columbia University, Code P (Patent)
  • Footnotes
    Support  Rejuvitas, Inc; International Retinal Research Foundation (IRRF); New York Stem Cell Foundation (NYSCF)
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3843. doi:
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      Joel Alan Imventarza, Keith Theodore, Bruna Lopes da Costa, Salvatore Marco Caruso, Stephen H. Tsang, Yi-Ting Tsai, Peter M.J. Quinn; Development of a Gene Augmentation Strategy for CRB1-mediated Inherited Retinal Diseases. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3843.

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

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Abstract

Purpose : Mutations in Crumbs homologue-1 (CRB1) lead to a diverse spectrum of inherited retinal diseases (IRDs). Currently, there is no treatment for patients with CRB1-linked IRDs. There are three abundantly expressed CRB1 isoforms in the human retina: CRB1-A, CRB1-C, and the recently identified CRB1-B. Mutations in CRB1 affect more than a single isoform, with the majority of mutations concurrently affecting CRB1-A and CRB1-B isoforms. In this study, we explore CRB1 isoform diversity and localization in human cadaveric retina. Further, using gene augmentation, we evaluate whether the overexpression of CRB1-A or CRB1-B alone or concomitantly in their predominantly cell-type-specific localizations will preserve retinal structure and function.

Methods : We performed RNA In-situ hybridization using CRB1 isoform-specific probes in human cadaveric retina. In parallel, by employing qPCR we assessed CRB1 isoform expression levels. Further, using the subretinal injection route, we assessed AAV-mediated delivery of codon-optimized CRB1-A or CRB1-B cDNAs alone or concomitantly in a Crb1 mouse model. We analyzed the eyes by histology, immunohistochemistry and electroretinography.

Results : We found by qPCR that CRB1-B was more highly expressed than CRB1-A or CRB1-C in the human cadaveric retina. In-situ hybridization on human cadaveric retina, using CRB1 isoform-specific probes, identified that CRB1-A transcript was localized predominately to the inner nuclear layer (INL) but some expression was found in the outer nuclear layer (ONL). CRB1-B predominately localized to the ONL and photoreceptor segments (PS), with occasional events seen in the INL. CRB1-C appeared more evenly disturbed between the ONL, PS and INL. Based on these findings, we hypothesize that the overexpression of CRB1-A and CRB1-B alone or concomitantly in their predominant cell-type-specific localizations could recover retinal structure and function. Our findings show that concomitant expression of CRB1-A and CRB1-B ameliorated retinal dysfunction in a Crb1 mouse model.

Conclusions : For the first time, we show the differences in localization of all three CRB1 isoforms in human cadaveric retina. CRB1-A and CRB1-B predominately localized to different cell types, Müller glial cells and photoreceptors, respectively. Lastly, we build upon previous pre-clinical gene therapy studies for CRB1-linked IRDs using CRB1-A and CRB1-B gene augmentation vectors.

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

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