Allele-specific gene editing approach for vision loss restoration in RHO-associated Retinitis Pigmentosa

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Abstract

Mutant RHO is the most frequent genetic cause of autosomal dominant retinitis pigmentosa. Here, we developed an allele-specific gene editing therapeutic drug to selectively target the human T17M RHO mutant allele while leaving the wild-type RHO allele intact for the first time. We identified a Staphylococcus aureus Cas9 (SaCas9) guide RNA that was highly active and specific to the human T17M RHO allele. In vitro experiments using HEK293T cells and patient-specific induced pluripotent stem cells (iPSCs) demonstrated active nuclease activity and high specificity. Subretinal delivery of a single adeno-associated virus serotype 2/8 packaging SaCas9 and sgRNA to the retinas of the RHO humanized mice showed that this therapeutic drug targeted the mutant allele selectively, thereby downregulating the mutant RHO mRNA expression. Administration of this therapeutic drug resulted in a long-term (up to 11 months after treatment) improvement of retinal function and preservation of photoreceptors in the mutant humanized heterozygous mice. Our study demonstrated a dose-dependent therapeutic effect in vivo. Unwanted off-target effects were not observed at the whole-genome sequencing level. Our study provides strong support for the further development of this effective therapeutic drug to treat RHO-T17M associated autosomal dominant retinitis pigmentosa (adRP), also offers a generalizable framework for developing gene editing medicine. Furthermore, our success in restoring the vision loss in the suffering RHO humanized mice verifies the feasibility of allele-specific CRISPR/Cas9-based medicines for other autosomal dominant inherited retinal dystrophies.

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All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided in Source Data 1, 2 and 3.

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Xiaozhen Liu

Department of Ophthalmology, Peking University Third Hospital, Beijing, China

Competing interests
No competing interests declared.
Jing Qiao

Beijing Chinagene Co, Ltd, Beijing, China

Competing interests
Jing Qiao, is an employee of Beijing Chinagene Co.,LTD and was employeed by Beijing Chinagene Co.,LTD at the time this work was conducted. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
Ruixuan Jia

Department of Ophthalmology, Peking University Third Hospital, Beijing, China

Competing interests
No competing interests declared.
Fan Zhang

Beijing Chinagene Co, Ltd, Beijing, China

Competing interests
Fan Zhang, is an employee of Beijing Chinagene Co.,LTD and was employeed by Beijing Chinagene Co.,LTD at the time this work was conducted. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed..
Xiang Meng

Department of Ophthalmology, Peking University Third Hospital, Beijing, China

Competing interests
No competing interests declared.
Yang Li

Beijing Tongren Eye Center, Capital Medical University, Beijing, China

Competing interests
No competing interests declared.
Liping Yang

Department of Ophthalmology, Peking University Third Hospital, Beijing, China

For correspondence
alexlipingyang@bjmu.edu.cn

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-4239-228X

Funding

the National Natural Science Foundation of China (81770966)

Liping Yang

the Beijing Natural Science Foundation of China (19JCZDJC64000(Z))

Liping Yang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Approval was obtained from the Peking University Health Science Center Ethics Committee for Experimental Animal Research (Research License LA20200473). All procedures were performed according to the regulations of the Association for Research in Vision and Ophthalmology's statement for the use of animals in ophthalmic and vision research. All mice were maintained in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care.Four adult, female NHP were bred and maintained at JOINN Laboratories (Suzhou, China), approval was obtained from the JOINN Laboratories Ethics Committee for Experimental Animal Research (Research License ACU21-1108).

Human subjects: The study was approved by the Medical Scientific Research Ethics Committee of Peking University Third Hospital (Research License 2021262). The procedures were performed in accordance with the tenets set forth in the Declaration of Helsinki. All patients provided written informed consent for this study.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.