Fig. 4: Adapting SLED vectors for translational studies.
From: Cell-specific regulation of gene expression using splicing-dependent frameshifting
a Diagramatic sketch of GFP under the control of the constitutive CMV promoter (CMV.GFP), PRPH2 under the control of the photoreceptor specific mOps promoter (mOps.PRPH2), and PRPH2 regulated by the photoreceptor-specific SLED.RAB (SLED.PRPH2) vectors designed for AAV packaging. CMV.GFP, mOps.PRPH2, and SLED.PRPH2 were packaged into AAV2.7m8 for testing in Prph2rds/rds animals. For experimental design, n = 6 for each AAV treatment. b Average ONL/INL ratios (Fig. S8) and c average light-adapted ERG b-wave amplitudes in three month post-injected Prph2rds/rds animals treated with CMV.GFP, SLED.PRPH2 or mOps.PRPH2 viral constructs (asterisks indicate p < 0.05, two-tailed t-test, comparison between mOps.PRPH2 and SLED.PRPH2, Fig. S8). d–f Immunofluorescence staining of retinal sections from Prph2rds/rds eyes injected with CMV.GFP (d), mOps.PRPH2 (e), and SLED.PRPH2 (f). ONL = outer nuclear layer, INL = inner nuclear layer. EGFP is only detected in CMV.GFP treated controls, but Prph2 signal (yellow arrow) is detected in photoreceptor inner segments of retinas transduced with both mOps.PRPH2 and SLED.PRPH2. g UCSC genome browser view of a cryptic exon in UBA1 (green arrow) that is present in cancers with oncogenic SF3B1 mutations (TCGA91) and absent in all normal human tissues sequenced by the GTEx consortium92. h Diagramatic sketch of bichromatic fluorescent reporter based on the SF3B1mut-associated exon (SLED.SFUv1) and a similar vector where an inducible iCaspase9 kill switch is coupled to incorporation of the SF3B1mut-associated exon. i As a proof of concept, SLED.SFUv1 was transfected into uveal melanoma cell lines with (Mel-202) and without (92-1) SF3B1 mutations. EGFP was highly enriched in only Mel-202 cells while dsRed was strongly expressed in 92-1 cells, which was validated using FACS (j). Isogenic cell lines derived from Mel-202 with the SF3B1R625G mutation genetically inactivated (PC76B6) and maintained (MR5) showed similarly concordance, with strong EGFP expression only present in the SF3B1R625G MR5 cell line. Likewise, strong EGFP expression was only present in SF3B1K700E K562 leukemia cells, as compared to wildtype K562 cells. ****p < 0.0001, two-tailed t-test. For ratio calculations, n = 245 (92-1), n = 1158 (Mel-202), n = 377 (PC76B6), n = 526 (MR5), n = 49 (K562WT), n = 677 (K562MUT). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. k Transfection of wildtype K562 cells and mutant SF3B1K700E K562 cells with SLED.SFUv2 revealed strong apoptosis only in SF3B1K700E K562 cells treated with the iCaspase9 activating dimerizer (n = 3 FACS replicates). Error bars in b, c, and k indicate standard deviation. Scale bars = 50 μm. ****p < 0.0001, two-tailed t-test. Source data are provided as a Source Data file.
