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Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9

Research output: Contribution to journalArticlepeer-review

Jiahui Wu, Oliver H. Bell, David A. Copland, Alison Young, John R. Pooley, Ryea Maswood, Rachel S. Evans, Peng Tee Khaw, Robin R. Ali, Andrew D. Dick, Colin J. Chu

Original languageEnglish
Pages (from-to)820-829
Number of pages10
JournalMolecular Therapy
Volume28
Issue number3
DOIs
Published4 Mar 2020

Bibliographical note

Funding Information: This work was supported by grant funding from the National Eye Research Centre ( BRI 019 ), the T.F.C. Frost Charitable Trust , Above & Beyond , Bristol , Fight for Sight ( 1730/1731 ), the Elizabeth Blackwell Institute for Health Research , the University of Bristol , and the Medical Research Council . Support was provided to P.T.K., R.R.A., and A.D.D. by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology . R.R.A. was also supported by Retina UK. C.J.C. was supported as an NIHR Academic Clinical Lecturer. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. A patent relating to this work has been filed by the University of Bristol. We wish to acknowledge the Wolfson Bioimaging Facility and the Genomics Facility at the University of Bristol for their assistance. The B6-RPE07 cell line was a gift of Dr. Heping Xu, Queen’s University Belfast, Belfast, UK. Shh10 plasmid was a gift from John Flannery & David Schaffer (Addgene plasmid #64867). pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA plasmid was a gift from Feng Zhang (Addgene plasmid #61591). 22 Funding Information: This work was supported by grant funding from the National Eye Research Centre (BRI 019), the T.F.C. Frost Charitable Trust, Above & Beyond, Bristol, Fight for Sight (1730/1731), the Elizabeth Blackwell Institute for Health Research, the University of Bristol, and the Medical Research Council. Support was provided to P.T.K. R.R.A. and A.D.D. by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. R.R.A. was also supported by Retina UK. C.J.C. was supported as an NIHR Academic Clinical Lecturer. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. A patent relating to this work has been filed by the University of Bristol. We wish to acknowledge the Wolfson Bioimaging Facility and the Genomics Facility at the University of Bristol for their assistance. The B6-RPE07 cell line was a gift of Dr. Heping Xu, Queen's University Belfast, Belfast, UK. Shh10 plasmid was a gift from John Flannery & David Schaffer (Addgene plasmid #64867). pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA plasmid was a gift from Feng Zhang (Addgene plasmid #61591).22 Publisher Copyright: © 2020 The Author(s) Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

King's Authors

Abstract

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.

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