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

  1. Xiaozhen Liu
  2. Jing Qiao
  3. Ruixuan Jia
  4. Fan Zhang
  5. Xiang Meng
  6. Yang Li
  7. Liping Yang  Is a corresponding author
  1. Peking University Third Hospital, China
  2. Beijing Chinagene Co, Ltd, China
  3. Capital Medical University, China

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.

Data availability

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.

Article and author information

Author details

  1. Xiaozhen Liu

    Department of Ophthalmology, Peking University Third Hospital, Beijing, China
    Competing interests
    No competing interests declared.
  2. 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..
  3. Ruixuan Jia

    Department of Ophthalmology, Peking University Third Hospital, Beijing, China
    Competing interests
    No competing interests declared.
  4. 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..
  5. Xiang Meng

    Department of Ophthalmology, Peking University Third Hospital, Beijing, China
    Competing interests
    No competing interests declared.
  6. Yang Li

    Beijing Tongren Eye Center, Capital Medical University, Beijing, China
    Competing interests
    No competing interests declared.
  7. Liping Yang

    Department of Ophthalmology, Peking University Third Hospital, Beijing, China
    For correspondence
    alexlipingyang@bjmu.edu.cn
    Competing interests
    No competing interests declared.
    ORCID icon "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.

Reviewing Editor

  1. Zhongjie Fu, Boston Children's Hospital, United States

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.

Version history

  1. Received: October 10, 2022
  2. Preprint posted: November 16, 2022 (view preprint)
  3. Accepted: June 2, 2023
  4. Accepted Manuscript published: June 5, 2023 (version 1)
  5. Version of Record published: June 19, 2023 (version 2)

Copyright

© 2023, Liu et al.

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.

Metrics

  • 2,195
    views
  • 296
    downloads
  • 3
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Xiaozhen Liu
  2. Jing Qiao
  3. Ruixuan Jia
  4. Fan Zhang
  5. Xiang Meng
  6. Yang Li
  7. Liping Yang
(2023)
Allele-specific gene editing approach for vision loss restoration in RHO-associated Retinitis Pigmentosa
eLife 12:e84065.
https://doi.org/10.7554/eLife.84065

Share this article

https://doi.org/10.7554/eLife.84065

Further reading

    1. Computational and Systems Biology
    2. Genetics and Genomics
    Ardalan Naseri, Degui Zhi, Shaojie Zhang
    Research Article Updated

    Runs-of-homozygosity (ROH) segments, contiguous homozygous regions in a genome were traditionally linked to families and inbred populations. However, a growing literature suggests that ROHs are ubiquitous in outbred populations. Still, most existing genetic studies of ROH in populations are limited to aggregated ROH content across the genome, which does not offer the resolution for mapping causal loci. This limitation is mainly due to a lack of methods for the efficient identification of shared ROH diplotypes. Here, we present a new method, ROH-DICE (runs-of-homozygous diplotype cluster enumerator), to find large ROH diplotype clusters, sufficiently long ROHs shared by a sufficient number of individuals, in large cohorts. ROH-DICE identified over 1 million ROH diplotypes that span over 100 single nucleotide polymorphisms (SNPs) and are shared by more than 100 UK Biobank participants. Moreover, we found significant associations of clustered ROH diplotypes across the genome with various self-reported diseases, with the strongest associations found between the extended human leukocyte antigen (HLA) region and autoimmune disorders. We found an association between a diplotype covering the homeostatic iron regulator (HFE) gene and hemochromatosis, even though the well-known causal SNP was not directly genotyped or imputed. Using a genome-wide scan, we identified a putative association between carriers of an ROH diplotype in chromosome 4 and an increase in mortality among COVID-19 patients (p-value = 1.82 × 10−11). In summary, our ROH-DICE method, by calling out large ROH diplotypes in a large outbred population, enables further population genetics into the demographic history of large populations. More importantly, our method enables a new genome-wide mapping approach for finding disease-causing loci with multi-marker recessive effects at a population scale.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Lisa Baumgartner, Jonathan J Ipsaro ... Julius Brennecke
    Research Advance

    Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino’s chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.