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<p>Retinitis pigmentosa (RP) associated with mutations in the rhodopsin gene (<i>RHO</i>) is a significant cause of blindness. Here we report on the application of adenine base editing of the c.1030C&gt;T (p.Q344X) <i>RHO</i> 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&gt;T <i>RHO</i> mutation and provide the foundation for base editing as a treatment for RP.</p><p></p>

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

  • 期刊论文

  • 发表日期：

    2025年12月10日

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  • Victoria A. C. Palmgren,
  • Miffy Hok Yan Cheng,
  • Yao Zhang,
  • Tiffany Carlaw,
  • Tessa Morin,
  • Jerry Leung,
  • Colin JD Ross,
  • Pieter R. Cullis,
  • Robert S. Molday

摘要

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.