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<p>Gene augmentation for inherited retinal diseases (IRDs) is constrained by the limited packaging capacity of viral vectors, rendering large genes such as <i>EYS</i> inaccessible to conventional delivery platforms. Here, we demonstrate a non-viral lipid nanoparticle (LNP) strategy for the intracellular delivery and transcription of full-length <i>EYS</i> plasmid DNA (~ 11.6 kb), a gene implicated in autosomal recessive retinitis pigmentosa. An LNP formulation based on the ionizable lipid KC2 and DOPC was first optimized using an eGFP reporter construct, achieving high encapsulation efficiency and robust transgene expression in ARPE-19 cells. The same formulation encapsulating the <i>EYS</i> construct showed single-copy loading and structural stability in human vitreous, while confocal imaging and RT-qPCR verified functional delivery in retinal pigment epithelial (RPE) cell cultures. In an ex vivo bovine retinal explant, laser capture microdissection followed by RT-qPCR of the photoreceptor layer confirmed <i>EYS</i> mRNA expression after mimicking subretinal injection. This provides direct evidence of nuclear plasmid delivery in retinal tissue, which closely preserves the laminar structure and cellular composition of the human retina. To our knowledge, this is the first report of in vitro<i>/</i>ex vivo LNP-mediated delivery of a large (&gt; 10 kbp) full-length IRD-relevant gene, underscoring the potential of LNPs for overcoming the size constraints of current gene therapies.</p> Graphical Abstract <p></p>

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Overcoming size barriers in retinal gene therapy via lipid nanoparticle-mediated delivery of full-length eys DNA

  • 期刊论文

  • 发表日期：

    2026年1月28日

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  • Sarah Goddaer,
  • Emma De Coster,
  • Elise Callens,
  • Emma Van Caenegem,
  • Olivier Van den Heule,
  • Rob W. J. Collin,
  • Karen Peynshaert,
  • Filip Van Nieuwerburgh,
  • Kevin Braeckmans,
  • Katrien Remaut

摘要

Gene augmentation for inherited retinal diseases (IRDs) is constrained by the limited packaging capacity of viral vectors, rendering large genes such as EYS inaccessible to conventional delivery platforms. Here, we demonstrate a non-viral lipid nanoparticle (LNP) strategy for the intracellular delivery and transcription of full-length EYS plasmid DNA (~ 11.6 kb), a gene implicated in autosomal recessive retinitis pigmentosa. An LNP formulation based on the ionizable lipid KC2 and DOPC was first optimized using an eGFP reporter construct, achieving high encapsulation efficiency and robust transgene expression in ARPE-19 cells. The same formulation encapsulating the EYS construct showed single-copy loading and structural stability in human vitreous, while confocal imaging and RT-qPCR verified functional delivery in retinal pigment epithelial (RPE) cell cultures. In an ex vivo bovine retinal explant, laser capture microdissection followed by RT-qPCR of the photoreceptor layer confirmed EYS mRNA expression after mimicking subretinal injection. This provides direct evidence of nuclear plasmid delivery in retinal tissue, which closely preserves the laminar structure and cellular composition of the human retina. To our knowledge, this is the first report of in vitro/ex vivo LNP-mediated delivery of a large (> 10 kbp) full-length IRD-relevant gene, underscoring the potential of LNPs for overcoming the size constraints of current gene therapies.

Graphical Abstract