So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.
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RNA-seq datasets are archived in the NCBI Gene Expression Omnibus under accession number GSE176527.
The R code to characterize novel LMI070-induced splicing events from RNA-seq data is available at https://github.com/DavidsonLabCHOP/Xon.
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We thank E. Lysenko, Z. Hilfinger and J. Weber of the Davidson laboratory, as well as N. Hoque of the Novartis Institute for Biomedical Research for technical assistance. This work was supported in part by the Hereditary Disease Foundation (A.M.M. and P.T.R.), T32 HG009495 (National Institutes of Health) (P.T.R.), NBIR (R.S., D.L., A.M.M. and B.L.D.) and the Children’s Hospital of Philadelphia Research Institute.
B.L.D. serves on the advisory boards and/or has sponsored research from Homology Medicines, Intellia Therapeutics, Spirovant Sciences, Resilience Bio, Patch Bio, Saliogen, Moment Bio, Triplet Biosciences, Panorama Medicines, Roche and NBIR. A.M.M. has sponsored research from NBIR.
Peer review information Nature thanks Alessandra Biffi, Mikhail Shapiro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Cartoon depicting SMN2 exon 7 in its native sequence or with splice-site modifications introduced to reduce background levels of exon 7 inclusion (3′ acceptor splice site, indSMN2). b, Representative RT–PCR reaction showing exon 7 inclusion with the SMN2-on cassettes in the absence of LMI070. The quantification of the exon 7 spliced-in or -out transcripts is depicted as the mean ± s.e.m. of 6 biological replicates. c, Exon 7 splicing of the SMN2 and indSMN2 cassette in response to LMI070 or RG7800. Representative RT–PCR reaction showing exon 7 inclusion as a function of LMI070 or RG7800 dose. The quantification of the exon 7 spliced-in or spliced-out transcripts are the relative transcript levels presented as the mean ± s.e.m. of 8 biological replicates. d, Luciferase activity of the SMN2 and indSMN2 cassettes in response to LMI070. The graph shows relative expression of firefly luciferase expressed from the SMN2-on or indSMN2-on cassettes in cells treated with DMSO or LMI070 (100 nM). The activity of the transfection control Renilla luciferase cassette is represented as a line above the bar graph. Data are the mean ± s.e.m. of 8 biological replicates.
Extended Data Fig. 2 Comparison between induced splice junctions in a previous study and in this work.
See ref. 8. a, Sequence logo of U1 RNA site targeted by LMI070 from 45 spliced-in exons identified by RNA-seq. b, To identify splice junctions with the highest induction across both studies we plotted the mean splice junction counts from our study against the mean splice junction counts from the dataset in ref. 8 reprocessed using our pipeline. The datasets correlate with Pearson’s r = 0.7, indicating that induced junctions performed similarly by rank across both datasets. Additionally, the top ranked splice junctions associated with SF3B3 and BECN1 were consistent across both datasets. c, Sashimi plots depicting novel LMI070-spliced in exons for the SF3B3 and BECN1 genes identified in our study and in ref. 8 by RNA-seq. Genomic location, position of the LMI070 spliced in exon, and intronic counts observed are indicated.
Extended Data Fig. 3 Candidate minigene cassette responsiveness to LMI070 and splicing response to LMI070 of the SF3B3-Xon cassette.
a, Luciferase induction of the minigene cassettes for SF3B3, BECN1, C12ORF4 and PDXDC2. The fold-change luciferase activity in LMI070-treated samples (depicted as +) is relative to DMSO-treated (depicted as −) cells, with data normalized to Renilla luciferase expression. Data are the mean ± s.e.m. of 8 biological replicates. ****P < 0.0001 for SF3B3 versus other candidate exons, ***P < 0.001 C12orf4 versus PDXDC2, one-way ANOVA with Bonferroni’s post hoc test. b, Luciferase activity of the minigene cassettes for SF3B3, BECN1, C12ORF4 and PDXDC2. Data show expression of firefly luciferase from the minigenes in response to DMSO (−) or LMI070 (+) treatment relative to Renilla luciferase activity. Data are mean ± s.e.m. of 8 biological replicates. c, Splicing analysis of the SF3B3-Xon cassette. Representative RT–PCR splicing assay (6 biological replicates) showing inclusion of the LMI070-induced SF3B3 exon in response to DMSO or LMI070 treatment. Inclusion of the LMI070 spliced-in exon was detected using primers binding the exons flanking the LMI070-induced exon (left), or using primers binding within the novel exon sequence (right).
Extended Data Fig. 4 Depiction of the frequency that non-AUG start codons are used determined by ribosome profiling.
a, Translation frequency from AUG and non-AUG start codons determined from ribosome profiling39. b, Depiction of non-AUG start codons in frame luciferase transcripts expressed under control of the SF3B3, BECN1, C12orf4 or PDXDC2 minigenes.
a, Firefly luciferase of the Xon cassettes in response to varying doses of LMI070 relative to Renilla luciferase (grey line). The data are the mean ± s.e.m. of 8 biological replicates. b, Representative RT−PCR for assessment of LMI070-induced pseudo exon expression. Exon inclusion in the Xon cassette was detected using primers flanking the pseudo exon. Data are the mean ± s.e.m. of 8 biological replicates. c, Representative gels from RT−PCR analysis for assessment of the LMI070-induced pseudo exons expressed from the noted promoters in response to varying doses of LMI070. Pseudo exon inclusion was detected using primers binding within the LMI070-induced pseudo exon and the downstream exon. Splicing was quantified and transcript levels presented as the mean ± s.e.m. of 8 biological replicates.
Extended Data Fig. 6 Assessment of the SF3B3-Xon cassette to control translation of eGFP in vitro and in vivo.
a, eGFP expression in HEK293 cells transfected with the SF3B3 minigene cassette (SF3B3-Xon-eGFP) and treated 24 h later with DMSO (left) or LMI070 (right). Bright-field panels are shown below (4 technical replicates). b, Representative photomicrograph of heart tissue sections showing eGFP in heart 24 h after treatment with LMI070 at 50 mg kg−1 (n = 2 mice). Scale bar, 200 μm. Inset, eGFP expression in cardiomyocytes at a higher magnification. Scale bar, 50 μm. c, Extended exposure of the western blot from Fig. 2c (4 mice per group). d, PCR assay demonstrates splicing activity in liver, heart and skeletal muscle in response to LMI070 (4 mice per group). e, Cartoon depiction of the Xon assays designed to quantify the LMI070-induced transcripts and eGFP expression levels from the Xon cassette after AAV9-Xon-eGFP gene transfer. f, Data show average Ct values for eGFP or LMI070-induced expression in heart and skeletal muscle. Fold change of the spliced-in expression cassette is shown relative to basal levels in mice injected with AAV9-Xon-eGFP and treated with vehicle (4 mice per group). g, Extended exposure of the western blot from Fig. 2h (3 mice per group). h, PCR assay demonstrates splicing activity in liver, heart and skeletal muscle after each LMI070 dose (3 mice per group). i, Data show average Ct values for eGFP or LMI070-induced expression in heart and skeletal muscle after each dose. Fold change of the spliced expression cassette is shown relative to basal levels in mice injected with AAV9-Xon-eGFP and treated with vehicle (3 mice per group).
a, Representative photomicrographs (5 mice per group) showing eGFP expression from mice treated intravenously 4 weeks earlier with AAVPHPeB-Xon-eGFP, and 24 h after treatment with vehicle or LMI070 at 5 or 50 mg kg−1. Thalamus (Th), hippocampus (Hc), cerebellum (Cb) and facial motor nucleus (VII), cortex (Cx), striatum (Str), substantia nigra (SN), and medial vestibular nucleus (MV) are shown. Scale bar, 100 μm; inset scale bar, 25 μm. In the hippocampus, * and ** denote the polymorphic and CA3 areas, respectively. In the cerebellum, * denotes the deep cerebellar nuclei. b, Splicing assays for exon inclusion in the cortex and the hippocampus of mice injected with AAVPHPeB-Xon-eGFP (3 mice per group). c, RT–qPCR of human progranulin expression. Data are mean ± s.e.m. of 5 mice per group, ****P < 0.0001 vehicle versus AAV-treated groups, one-way ANOVA followed by Bonferroni’s post-hoc test. d, Splicing assays for exon inclusion in cortex samples of mice injected with AAVPHPeB-Xon-PGRN (3 mice per group).
Extended Data Fig. 8 Generation of the miniXon cassette and assessment of miniXon control of SaCas9 for in vivo gene editing in liver.
a, Cartoon depicting the AAV genome size with the SF3B3-Xon and SF3B3-miniXon cassettes. b, Luciferase induction in HEK293 cells transfected with SF3B3-miniXon-luciferase or SF3B3-Xon-luciferase in response to varying doses of LMI070. All samples are normalized to Renilla luciferase activity and are relative to DMSO treated cells. Data are the mean ± s.e.m. of 8 biological replicates (***P < 0.001 versus SF3B3.Xon, two-way ANOVA followed by Bonferroni’s post hoc test). c, Splicing inclusion assays of the LMI070-induced exon at 100 nM LMI070. Pseudo exon inclusion in the Xon cassette was detected using primers flanking the pseudoexon (left) or by priming within the novel exon sequence (right; 4 technical replicates). d, Experimental design. Mice were injected with AAV8-miniXon-SaCas9 plus AAV8-sgAi14-eGFP (1 × 1012 viral genomes, 1:1 ratio) and 2 weeks later dosed with vehicle or LMI070 at 50 mg kg−1 to induce SaCas9 expression and editing of the loxP-STOP cassette (guides: sgA14_1: 5′-CTCTAGAGTCGCAGATCCTC-3′, sgAi14_2: 5′-ACGAAGTTATATTAAGGGTT-3′). One week later, mice were euthanized, and livers processed to assess gene editing by genomic DNA PCR, histology and FACS of isolated hepatocytes. e, Representative FACS analysis of hepatocytes obtained from Ai14 mice after LMI070 or vehicle treatment. The gating/sorting strategy (above), and the percentage of tdTomato expressing cells for each condition (below) is shown (4 biological replicates). f, Representative photomicrographs of liver sections obtained from AAV injected Ai14 mice 1-week after LMI070 treatment. tdTomato expression (red) is evident in LMI070 treated mice (5 mice per group). Scale bars, 100 μm. g, SaCas9 mediated editing of the loxP-STOP cassette in Ai14 mice as detected by PCR assay of liver genomic DNA (3 of 5 mice with guides plus LMI, 2 of 4 mice with guides plus vehicle, 1 of 2 untreated mice are shown). A PCR product of 355 bp size corresponding to the edited Ai14 ROSA Locus was observed in the LMI070-treated mice. h, Sanger sequencing of the 355 bp PCR product confirmed targeted deletion of the loxP-STOP cassette and DNA repair of the Ai14 reporter locus.
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Monteys, A.M., Hundley, A.A., Ranum, P.T. et al. Regulated control of gene therapies by drug-induced splicing. Nature 596, 291–295 (2021). https://doi.org/10.1038/s41586-021-03770-2