Development of tools to induce exon skipping of a cryptic exon driving HLRCC
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1238264 | ENA |
REPOSITORIES: ENA
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Project description:Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) arises from the loss of fumarate hydratase (FH) activity, leading to the development of cutaneous and uterine leiomyomas as well as early-onset type 2 papillary renal cell carcinoma. Recently, we identified a pathogenic intronic variant in the FH gene that disrupts splicing by creating a novel splice acceptor site, resulting in the aberrant inclusion of a cryptic exon. This exon introduces a premature termination codon, leading to loss of enzymatic function. To restore FH expression, we aimed to identify strategies to exclude the cryptic exon from the mature mRNA. To this end, we generated a mini-gene GFP reporter system that recapitulates the splicing defect observed in patients. We employed CRISPR-Cas9-mediated genome editing and antisense oligonucleotides (ASOs) to modulate splicing and were able to show that both strategies can successfully promoted exon skipping in a reporter cell line. Furthermore, we were able to shift the balance between the different FH mRNA isoforms in patient derived fibroblasts using ASOs. These findings support the potential of splicing modulation as a therapeutic approach for HLRCC-associated FH non-coding loss-of-function mutations.
2025-08-19 | GSE292376 | GEO
Project description: Not available
| S-EPMC12396411 | biostudies-literature
Project description:Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular and cellular biology, medicine and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings preventing their broader implementation including transient effects in the case of oligonucleotides or limiting PAM motifs, sequence context preferences for deaminases, and undesirable cryptic splicing in the case of gene editing tools. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused with different deaminases for simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing not only improves exon skipping rates but also reduces aberrant outcomes such as cryptic splicing and intron retention. SPLICER enables editing of exon splice sites with high efficiency, including many exons refractory to splicing reprogramming by the native SpCas9 BEs. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which contains the amino acid residues responsible for the formation of Aβ plaques in Alzheimer’s disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aβ42 peptides in vitro while inducing DNA editing and exon skipping in vivo within a humanized mouse model of Alzheimer’s disease. Overall, SPLICER is a widely applicable and highly efficient toolbox for exon skipping with broad therapeutic applications.
2023-12-10 | GSE246586 | GEO
Project description:Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular and cellular biology, medicine and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings preventing their broader implementation including transient effects in the case of oligonucleotides or limiting PAM motifs, sequence context preferences for deaminases, and undesirable cryptic splicing in the case of gene editing tools. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused with different deaminases for simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing not only improves exon skipping rates but also reduces aberrant outcomes such as cryptic splicing and intron retention. SPLICER enables editing of exon splice sites with high efficiency, including many exons refractory to splicing reprogramming by the native SpCas9 BEs. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which contains the amino acid residues responsible for the formation of Aβ plaques in Alzheimer’s disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aβ42 peptides in vitro while inducing DNA editing and exon skipping in vivo within a humanized mouse model of Alzheimer’s disease. Overall, SPLICER is a widely applicable and highly efficient toolbox for exon skipping with broad therapeutic applications.
2023-12-10 | GSE246587 | GEO
Project description:Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin, inhibit the interaction between EWS, an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene encoding the main p53 ubiquitin-ligase. This reversible exon skipping participates in the timely regulation of MDM2 expression, and may contribute to the accumulation of p53 during stress exposure and its rapid shut off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to camptothecin and EWS/YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, resulting in widespread exon skipping and playing a central role in the genotoxic stress response. 6 samples of MCF7 cells exposed to different treatments were analyzed: 3 x control_6 hours; 3 x camptothecin_6 hours.
2010-06-09 | E-GEOD-21840 | biostudies-arrayexpress
Project description:Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by loss of function mutations in the dystrophin gene. There is currently no cure for DMD although various promising approaches are progressing through human clinical trials. Among them, exon-skipping therapy using antisense oligonucleotides (AONs) aim to exclude a selected exon at one edge of the mutation to subsequently restore dystrophin production. To speed-up translation from the bench to bedside, we describe here the implementation of a fast and simple multi-phase funnel cascade to identify and triage tricyclo-DNA (tcDNA) AONs, notably for rare mutations such as mutation in the DMD exon 16. Using an innovative high-throughput screening solution to assess dystrophin protein restoration after AONs treatment and a panel of non-clinical safety assessments, we first selected from a 59 AONs walk, tc-5 as the most efficient candidate with the appropriate safety profile to exclude DMD exon 16. Preclinical in vivo investigations in mice and non-human primates define the pharmaco- kinetics/dynamics (PK, PD) properties and biodistribution profiles of tc-5, demonstrate efficient exon-16-skipping and highlight a particularly encouraging safety profile. To counteract the lack of study model to assess exon-16-skipping, we generated appropriate tri-dimensional (3D) human DMD disease model. In such engineered human DMD mEx16 “mini-muscle”, tc-5 treatment results in a dose-dependent exon-skipping and restoration of dystrophin protein. Subsequent transcriptomic and proteomic analyses demonstrated dose-dependent benefits at both levels highlighting a rescued ontogenesis by tc-DNA exon skipping-mediated dystrophin restoration. While providing the foundation for an expedited path towards preclinical evaluation of personalized AONs therapies, notably for rare genetic defects lacking dedicated preclinical disease model as well as therapeutic splicing solution, this work deciphers the relationship between AONs doses, exon-skipping efficacy, dystrophin restoration and benefits of tc-DNA on the ontogenesis in the appropriate human dystrophic context.
2026-09-14 | PXD060394 | Pride
Project description:Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently demonstrated that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of adenine-deaminase base editors to disrupt the conserved adenosine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9 nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress towards permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.
2019-09-12 | GSE120125 | GEO
Project description:we induce exon skipping and generate a gain-of-function of an oncogene, β-catenin, using CRISPR/Cas9 in mouse liver cells. Specifically, a single guide RNA (sgRNA) targeting exon 3 of β-catenin induces exon skipping and gain-of-function of β-catenin in mouse hepatocytes. In synergy with YAPS127A, exon skipped hepatocytes gain tumorigenic ability and are thus enriched via tumor formation. Surprisingly, characterization of the exon-skipped tumors reveals two distinct subtypes with different histological features. Remarkably, ectopic expression of two representative exon-skipped β-catenin transcripts together with YAPS127A phenocopies the two histologically distinct subtypes of liver cancer. Finally, the transcriptome sequencing analysis reveal two subtypes of liver cancer and most importantly, one subtype of the exon-skipped tumor shows features of hepatoblastoma, while the other does not. This exon skipping model reveals CRISPR/Cas9 can lead to exon-skipped transcripts with in frame coding and gain-of-functions.
2021-01-01 | GSE164109 | GEO
Project description:Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin, inhibit the interaction between EWS, an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene encoding the main p53 ubiquitin-ligase. This reversible exon skipping participates in the timely regulation of MDM2 expression, and may contribute to the accumulation of p53 during stress exposure and its rapid shut off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to camptothecin and EWS/YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, resulting in widespread exon skipping and playing a central role in the genotoxic stress response.
2010-05-20 | GSE21840 | GEO
Project description:Psychological stress reactions can stimulate mammalian immune functions due to yet unknown mechanisms. We hypothesized that these involve massive post-stress alternative splicing modulations in peripheral blood mononuclear cells (PBMCs). RNA was extracted from PBMCs of BALB/C mice following unpredictable repeated foot shocks. Among the tested group, five mice exhibiting the maximal circulation glucocorticoids were selected for the stress group. PBMC RNA of 5 BALB/C mice served as the control group. Through linear regression analysis of all the reciprocal junction pairs represented on the microarrays and the Ensembl database, 496 alternative splicing events were detected. The stressed mice showed 65% exon skipping out of total splicing event changes compared to controls. The detected genes exhibited functional enrichment (through DAVID EASE analysis) in alternative splicing (47%), cellular response to stress (12%), lymphocyte activation (8%), stress-induced proteins (2%) and heat-shock-induced proteins (2%). Specifically, exon skipping modifications in the Hnrnph1 and CLK1 splicing-related transcripts were accompanied by stress-inducible inclusions in the immune response-related IRF-1 gene. Our findings demonstrate dependence on exon skipping and independence from glucocorticoid and innate immunity for the stress-inducible exacerbation of immunity and open new venues for preventing post-trauma inflammatory crisis.
2018-04-15 | GSE37856 | GEO