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Mutant RHO is the most frequent genetic cause of autosomal dominant retinitis pigmentosa (adRP). 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 single guide RNA (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 heterozygous mutant humanized 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 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.
Pubmed ID: 37272616 RIS Download
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View all literature mentionsCurated database of known (published) gene lesions responsible for human inherited disease.
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View all literature mentionsA commercial antibody supplier which supplies primary and secondary antibodies, biochemicals, proteins, peptides, lysates, immunoassays and other kits.
View all literature mentionsAn online resource which helps researchers manage and organize labs and experimental results by supplying molecular biology software tools for experimental design and data analysis. Benchling provides tools for functions such as primer design and colony counting as well as CRISPR guide design and automated Gibson and Golden Gate cloning. Users can take notes in line with data, link data across entries, keep files and data in one place, and manage and keep track of team progress. An enterprise version of Benchling is available for scientists working within an organization with additional administrative, compliance, and security protocols.
View all literature mentionsWeb application that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Enables searching for potential off-target sites in any sequenced genome rapidly without limiting PAM sequence or number of mismatched bases.
View all literature mentionsCurated database of known (published) gene lesions responsible for human inherited disease.
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View all literature mentionsCell line HEK293T is a Transformed cell line with a species of origin Homo sapiens (Human)
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