Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids

Kamil Kruczek; Zepeng Qu; James Gentry; Benjamin R Fadl; Linn Gieser; Suja Hiriyanna; Zachary Batz; Mugdha Samant; Ananya Samanta; Colin J Chu; Laura Campello; Brian P Brooks; Zhijian Wu; Anand Swaroop

doi:10.1016/j.stemcr.2020.12.018

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids

Kamil Kruczek, Zepeng Qu, James Gentry, Benjamin R Fadl, Linn Gieser, Suja Hiriyanna, Zachary Batz, Mugdha Samant, Ananya Samanta, Colin J Chu, Laura Campello, Brian P Brooks, Zhijian Wu, Anand Swaroop

Bristol Medical School (THS)

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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Abstract

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

Original language	English
Pages (from-to)	252-263
Number of pages	12
Journal	Stem Cell Reports
Volume	16
Issue number	2
Early online date	28 Jan 2021
DOIs	https://doi.org/10.1016/j.stemcr.2020.12.018
Publication status	Published - 9 Feb 2021

Bibliographical note

Funding Information:
We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute ( ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases , National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster ( http://hpc.nih.gov ).

Funding Information:
We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute (ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster (http://hpc.nih.gov).

Publisher Copyright:
© 2020 The Authors

Keywords

pluripotent stem cells
iPSC
3-D organoids
retinal degeneration
AAV
therapy
disease modeling
transcription factor
transcriptome
scRNA-seq

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Kruczek, Kamil ; Qu, Zepeng ; Gentry, James ; Fadl, Benjamin R ; Gieser, Linn ; Hiriyanna, Suja ; Batz, Zachary ; Samant, Mugdha ; Samanta, Ananya ; Chu, Colin J ; Campello, Laura ; Brooks, Brian P ; Wu, Zhijian ; Swaroop, Anand. / Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids. In: Stem Cell Reports. 2021 ; Vol. 16, No. 2. pp. 252-263.

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title = "Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids",

abstract = "Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.",

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author = "Kamil Kruczek and Zepeng Qu and James Gentry and Fadl, {Benjamin R} and Linn Gieser and Suja Hiriyanna and Zachary Batz and Mugdha Samant and Ananya Samanta and Chu, {Colin J} and Laura Campello and Brooks, {Brian P} and Zhijian Wu and Anand Swaroop",

note = "Funding Information: We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute ( ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases , National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster ( http://hpc.nih.gov ). Funding Information: We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute (ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster (http://hpc.nih.gov). Publisher Copyright: {\textcopyright} 2020 The Authors",

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Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids. / Kruczek, Kamil; Qu, Zepeng; Gentry, James; Fadl, Benjamin R; Gieser, Linn; Hiriyanna, Suja; Batz, Zachary; Samant, Mugdha; Samanta, Ananya; Chu, Colin J; Campello, Laura; Brooks, Brian P; Wu, Zhijian; Swaroop, Anand.

In: Stem Cell Reports, Vol. 16, No. 2, 09.02.2021, p. 252-263.

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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AU - Qu, Zepeng

AU - Gentry, James

AU - Fadl, Benjamin R

AU - Gieser, Linn

AU - Hiriyanna, Suja

AU - Batz, Zachary

AU - Samant, Mugdha

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AU - Chu, Colin J

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AU - Brooks, Brian P

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N1 - Funding Information: We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute ( ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases , National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster ( http://hpc.nih.gov ). Funding Information: We are grateful to the patients and their families for tissue donations, which enabled this study. We thank Jeanette Beers at the NIH/National Heart, Lung, and Blood Institute iPS and Genome Engineering Core Facility for generating iPSC lines, Sandra Burkett at the Molecular Cytogenetics Core Facility, National Cancer Institute Center for Cancer Research for karyotyping, and Milton A. English and Charles Drinnan for fibroblast cell line generation and stem cell maintenance. This research was supported by the Intramural Research Programs of the National Eye Institute (ZIAEY000450 and ZIAEY000546 to A. Swaroop) and, in part, by the National Institute of Allergy and Infectious Diseases, National Institutes of Health. We are grateful to Opsomai Foundation (Italy) for a generous donation, which partly funded this work. This work used the high-performance computational capabilities of the NIH Biowulf Linux cluster (http://hpc.nih.gov). Publisher Copyright: © 2020 The Authors

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N2 - Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX-LCA and other CRX retinopathies.

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KW - pluripotent stem cells

KW - iPSC

KW - 3-D organoids

KW - retinal degeneration

KW - AAV

KW - therapy

KW - disease modeling

KW - transcription factor

KW - transcriptome

KW - scRNA-seq

U2 - 10.1016/j.stemcr.2020.12.018

DO - 10.1016/j.stemcr.2020.12.018

M3 - Article (Academic Journal)

C2 - 33513359

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SP - 252

EP - 263

JO - Stem Cell Reports

JF - Stem Cell Reports

SN - 2213-6711

IS - 2

ER -

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids

Abstract

Bibliographical note

Keywords

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