Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman
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Research Article Cell biology Genetics

Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme

Abstract

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus–based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Authors

Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman

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Figure 5

Effect of AAV-LCA5 gene augmentation therapy on distal axoneme organization and CC length.

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Effect of AAV-LCA5 gene augmentation therapy on distal axoneme organizat...
(A–D) Widefield (original magnification, 63×) images of expanded photoreceptors stained for tubulin (magenta) and lebercilin (LCA5; orange, A), CEP290 (cyan, B), POC5 (green, C), or RP1 (white/gray, D) from P18 to P28 in AAV-LCA5 gene therapy–treated Lca5gt/gt mice (HOM + Therapy). Lines in P18 LCA5 image (A) illustrate measurements shown in F of tubulin width at 3 locations: +500 nm, 0 nm, −500 nm. The proximal end of the LCA5 signal was used to set the 0 location. A indicates the percentage of photoreceptors that express LCA5 at each time point (n = 109–184). Three animals per time point. Scale bars: 500 nm. (E) Distal axoneme (above CC) conformations of HOM + Therapy photoreceptors from P18 to P28 indicated in percentages. Photoreceptor distal axoneme conformations: normal (87%), open/broken (9.6%), and bent/curled (3.4%). n = 146. (F) Tubulin width measurements of P18 HOM photoreceptors, gene therapy treated versus nontreated, at the 3 locations depicted in A. Average tubulin width at each location is indicated by a red or blue dot for HOM and HOM + Therapy, respectively. Only photoreceptors that express LCA5 were used for the measurements. HOM measurements correspond to the data presented in Figure 3C. Three animals per time point. Data presented as mean ± SD; n = 27–37. ****P < 0.0001 by F test. Significance represents tubulin width dispersion between HOM and HOM + Therapy. (G–I) Impact of AAV-LCA5 gene therapy on CEP290 length (G), CC inner scaffold length (POC5, H), or RP1-normalized intensity at the bulge region (I) from P18 to P28. HET and HOM measurements correspond to the data in Figure 3, G–I. Three animals per time point. Data presented as mean ± SD; n = 39–65 (G), n = 19–60 (H), n = 39–62 (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Kruskal-Wallis test with Dunn’s multiple-comparison test.