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. 2024 Oct 11;33(24):2094–2110. doi: 10.1093/hmg/ddae144

Figure 5.

Figure 5

Reconstitution of IFT140 by CRE-lox-mediated recombination. (A) Schematic representation of IFT140 reconstitution by CRE-lox-mediated recombination of bipartite AAV vector genomes. T2A: T2A “self-cleaving” peptide, IRES: Internal ribosome entry site. Others are the same as in Fig. 3. (B) Production of full-length IFT140 proteins (red arrowheads) through CRE-lox-mediated recombination in HEK293T cells. HEK293T cells were transduced with dual AAV vectors depicted in panel A (with a CMV promoter), and cell lysates were subjected to SDS-PAGE followed by immunoblotting with HA and IFT140-C antibodies. Lane 1: AAV-IFT140_N only, lane 2: AAV-IFT140_C only, lane 3: Dual AAV-IFT140_N+C. β-actin was used as a loading control. (C) Expression of full-length IFT140 in normal mouse retinas using the dual AAV-IFT140 CRE/lox set. Dual AAV-GRK1p-IFT140 vectors were administered via subretinal injection into mouse eyes (serotype: Anc80L65, dose: 8 × 108 GC per vector), and eyes were collected 2 weeks post-injection for immunoblotting with HA, IFT140-C, and CRE antibodies. Lanes 1–2: Uninjected (n = 2), lanes 3–5: AAV-IFT140_N only (n = 3), lanes 6–8: AAV-IFT140_C only (n = 3), and lanes 9–12: AAV-IFT140_N+C (n = 4). Each lane corresponds to an individual eye. The blue and black arrowheads indicate the IFT140-N product and CRE, respectively. (D) Reconstitution of IFT140 expression in Ift140 CKO mice using dual AAV-GRK1p-IFT140 vectors. Dual AAV-GRK1p-IFT140 CRE/lox vectors were delivered to the subretinal space of normal (Ift140fl/fl;iCre75−) and Ift140 CKO (Ift140fl/fl;iCre75+) mice at P16, and IFT140 expression was analyzed by immunoblotting at P55. Both injected and uninjected eyes were collected, and their protein extracts were loaded side-by-side (indicated by black lines). Each lane corresponds to an individual eye. (E and F) Preservation of photoreceptor cells in Ift140 CKO mice by dual AAV-GRK1p-IFT140 CRE/lox vectors. Dual AAV-GRK1p-IFT140 CRE/lox vectors were delivered to the subretinal space of normal and Ift140 CKO mice at P16, and retinal histology was examined by immunohistochemistry using HA, PRPH2, and GNAT2 antibodies at P52. Nuclei were counterstained with DAPI. Brackets delineate the outer nuclear layer (ONL). Scale bar: 50 μm. INL: Inner nuclear layer. (G and H) Preservation of retinal function in Ift140 CKO mice by IFT140 subretinal gene therapy. Normal (n = 6) and Ift140 CKO (n = 6) mice were administered dual AAV-GRK1p-IFT140 vectors in one eye at P16, and rod (G) and cone (H) functions were evaluated by scotopic and photopic ERG at P45. Uninjected contralateral eyes were used as controls. Rod function was measured using 0.01 cd⋅s/m2 dim flashes after dark adaptation, while cone function was measured using 3.0 cd⋅s/m2 bright flashes after light adaptation. ERG b-wave amplitudes (mean ± SD) are shown in the graphs. Asterisks indicate statistical significance (two-tailed Student’s t-test; P < 0.05). ns: not significant.