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Lipid nanoparticle mediated base editing of the Q344X rhodopsin mutation associated with retinitis pigmentosa

Gene Therapy (2025)Cite this article

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Abstract

Retinitis pigmentosa (RP) associated with mutations in the rhodopsin gene (RHO) is a significant cause of blindness. Here we report on the application of adenine base editing of the c.1030C>T (p.Q344X) RHO mutation linked to RP. Using a fluorescence reporter cell system, we optimized editing by exploring base editors, sgRNA, and delivery methods. Flow cytometry, western blotting, and immunofluorescence microscopy confirmed the restoration of full-length rhodopsin after editing. DNA sequencing verified editing at the target nucleotide and the absence of bystander edits within the editing window. Polyethylenimine cationic polymer transfection of cells with a plasmid containing the NG-ABE8e adenine base editor and A6 guide RNA that placed the targeted adenine in position 6 of the editing window resulted in 31.0% gDNA sequence correction and 26.3% rhodopsin protein correction as determined by flow cytometry. Purified NG-ABE8e protein complexed with A6-sgRNA showed 32.2% gDNA editing and 44.5% rhodopsin correction. Plasmid NG-ABE8e and A6-sgRNA co-encapsulated into lipid nanoparticles (LNPs) and transfected into the reporter cell system resulted in the highest editing (42.6% gDNA editing and 65.9% rhodopsin correction). These results demonstrate the successful correction of the c.1030C>T RHO mutation and provide the foundation for base editing as a treatment for RP.

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Fig. 1: Validation of Rho-EGFP and Rho-Q344X-EGFP stable cell lines.
Fig. 2: Base editor selection and sgRNA design for the Rho-Q344X-EGFP mutant.
Fig. 3: Editing of the Rho-Q344X mutation using PEI to transfect plasmid base editors.
Fig. 4: Editing of the Rho-Q344X mutation using plasmid ABE encapsulated in LNPs.
Fig. 5: Editing of Rho-Q344X mutation using Ribonucleoprotein (RNP).

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Data availability

All the data produced in this study have been either included in the published paper or are accessible through the lead contact upon request.

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Acknowledgements

We thank Dr. Jonathen Yen for the NG-ABE8e ribonucleoprotein expression plasmid and Dr. Orson Moritz for the murine rhodopsin cDNA. Ryan Zhu assisted in RNP purification. Cryo-TEM data was collected at the High-Resolution Macromolecular Electron Microscopy (HRMEM) facility at UBC supported by the Canadian Foundation of Innovation and the British Columbia Knowledge Development Fund.

Funding

This study was supported by the Canadian Institutes of Health Research (CIHR) grant 175118 and an unrestricted UBC research grant to RSM and CIHR grant 148469 to PRC. MHYC was supported by the NanoMedicines Innovation Network postdoctoral fellowship in gene therapy and the CIHR Research Excellence, Diversity, and Independence (REDI) Early Career Transition Award. Y.Z. (FBD 193487) and JL was supported by a Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award. VACP, YZ, and TM were awarded NanoMedicines Innovation Network (NMIN) graduate awards.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

    Victoria A. C. Palmgren, Yao Zhang, Jerry Leung, Pieter R. Cullis & Robert S. Molday

  2. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada

    Miffy Hok Yan Cheng, Tiffany Carlaw, Tessa Morin & Colin JD Ross

  3. School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada

    Yao Zhang & Tiffany Carlaw

  4. Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Yao Zhang

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  1. Victoria A. C. Palmgren
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Contributions

RSM and VACP conceived the overall project. VACP, MHYC, YZ, TC, TM, JL designed the experiments, acquired the data and along with RSM, CJDR, and PRC analyzed and interpreted the data. RSM, CJDR and PRC supervised the study and provided resources. VACP and RSM wrote the original draft of the manuscript. All authors reviewed, edited and approved the manuscript.

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Correspondence to Robert S. Molday.

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PRC has financial interests in Acuitas Therapeutics, Mesentech, and NanoVation Therapeutics. The other authors declare no competing interests.

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Palmgren, V.A.C., Cheng, M.H.Y., Zhang, Y. et al. Lipid nanoparticle mediated base editing of the Q344X rhodopsin mutation associated with retinitis pigmentosa. Gene Ther (2025). https://doi.org/10.1038/s41434-025-00584-z

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