Individual genetic polymorphisms can exert a modifier effect on splicing. With about one single-nucleotide polymorphism per 250 bases, we hypothesized that consideration of splice haplotypes will improve the accuracy of spliceogenic variant annotation, as current methods mainly assess single DNA variants and in a single reference genome. We describe Panthera for high-throughput pangenomic analysis of splice haplotypes, available as an open-source on GitHub (https://github.com/CherWeiYuan/Panthera). It accepts user-input variants and uses a deep learning model to predict their combinatorial effect on splice site probabilities under 64 non-redundant genetic backgrounds derived from five human super populations. The 874,587-parameter model is designed based on splicing mechanisms where multiple functional motifs, spliceosomal and auxiliary elements, interact with the sequential implementations of convolutional neural network (CNN) and Transformer blocks to simulate local and full-sequence motif interactions respectively. Panthera was validated to accurately detect every experimentally validated splice haplotype reported for FAS exon 6 and CFTR exon 10. Subsequently, two genes were used as case studies to demonstrate Panthera’s capability to identify novel splice haplotypes and novel splice modifiers therein. In MLH1, a haploinsufficient tumour suppressor whose loss of functional expression causes Lynch syndrome and colorectal cancers, Panthera identified a novel splice modifier in tandem with each of the two novel splice variants predicted to activate two respective novel frameshifting pseudoexons in an African individual. For the pathogenic PCCB c.654 + 462A > G splice variant implicated in propionic acidemia, Panthera identified a novel splice haplotype with a novel splice modifier, a CTGATGT insertion, that significantly enhances the pseudoexon activation in 55 genetic backgrounds. Notably, all predictions were experimentally validated through splicing assays on minigenes constructed with the respective splice haplotypes expressed in two cell lines. The case studies of FAS, CFTR, MLH1, and PCCB suggest haplotype differences can influence splicing, and a pangenomic haplotype tool is essential to detect them.

RAS family proteins, including HRAS, NRAS, and KRAS, are frequently mutated in cancer. Although there has been recent success in designing inhibitors that target oncogenic RAS, they elicit resistance and treating RAS-driven cancer remains difficult. Here, employing a proteomic analysis, we find that multiple spliceosome components are upregulated in the nuclei of cells undergoing RAS-induced senescence. This upregulation depends on RAS signalling and occurs in both senescent preneoplastic and fully transformed cancer cells. Spliceosome components are also highly expressed in preneoplastic and cancerous lesions in human and murine lung, liver, colorectal, and pancreatic cancers. Using siRNA screens, we identify six spliceosome components, including SF3B1 and RBM39, that are essential in cells expressing oncogenic RAS. We find that SF3B1 is required in these cells for maintaining splicing fidelity. By combining transcriptome and splicing analyses with functional screens, we identify the RNA Pol II-associated factor SPT5 as a key mediator of the SF3B1 effects. Importantly, using mouse models of liver cancer, we show that RBM39 and SF3B1 inhibitors are effective in targeting both preneoplastic lesions and aggressive tumours expressing oncogenic RAS. In summary, our study highlights the spliceosome as a promising target for RAS-driven cancers capable of inhibiting both cancer initiation and progression.

Nucleic acid therapeutics are used for silencing, expressing or editing genes in vivo. However, their systemic stability and targeted delivery to bone marrow resident cells remains a challenge. In this study we present a nanotechnology platform based on natural lipoproteins, designed for delivering small interfering RNA (siRNA), antisense oligonucleotides and messenger RNA to myeloid cells and haematopoietic stem and progenitor cells in the bone marrow. We developed a prototype apolipoprotein nanoparticle (aNP) that stably incorporates siRNA into its core. We then created a comprehensive library of aNP formulations and extensively characterized their physicochemical properties and in vitro performance. From this library, we selected eight representative aNP-siRNA formulations and evaluated their ability to silence lysosomal-associated membrane protein 1 (Lamp1) expression in immune cell subsets in mice after intravenous administration. Using the most effective aNP identified from the screening process, we tested the platform’s potential for therapeutic gene silencing in a syngeneic murine tumour model. We also demonstrated the aNP platform’s suitability for splice-switching with antisense oligonucleotides and for protein production with messenger RNA by myeloid progenitor cells in the bone marrow. Our data indicate that the aNP platform holds translational potential for delivering various types of nucleic acid therapeutics to myeloid cells and their progenitors. In this study, the authors present optimization and efficacy testing of apolipoprotein-based lipid nanoparticles for delivering various nucleic acid therapeutics in vivo to immune cells and their progenitors in the bone marrow.

The endogenous U1 small nuclear RNA (U1-snRNA) plays a crucial role in splicing initiation through base-pairing to donor splice sites (5'-SSs). Likewise, modified U1s that carry a mutation-adapted 5'-terminal sequence have been demonstrated to rescue exon splicing when this is disrupted by genetic mutations within the 5'-SS. Given the base-pairing flexibility of the endogenous U1, the selectivity of modified U1s requires investigation. We developed a computational pipeline (Utargetome) that considers combinations of mismatches and alternative annealing registers to predict the transcriptome-wide binding sites (or targetome) of a U1. The pipeline accuracy was tested by recapitulating well-established alternative annealing registers and specificity for 5'-SSs in the predicted targetome of the human endogenous U1. It was then applied to analyse the targetome of 54 modified U1s that have been demonstrated to restore exon inclusion when affected by 5'-SS pathogenic mutations. While the targetome size was found to be wide-ranging, the off-target load appeared to be reduced for U1s targeting distal sites from the canonical U1-binding position. This feature was predicted also for a large set of 30,204 newly designed U1s targeting 839 5'-SS pathogenic mutations that were expected to affect exon inclusion. Targetome analysis indeed revealed an optimal distal-targeting position at 3 nucleotides downstream from the canonical 5'-SS, for which a modified U1 is likely to have minimal off-targets at 5'-SSs and acceptor splice sites (3'-SSs). Based on these insights, we propose to implement targetome prediction in the design and optimization of therapeutic U1s with improved selectivity.

Chemical optimization of ribose has significantly advanced nucleic acid therapeutics (NATs) by improving the stability, specificity, and safety of therapies like small interfering RNAs, CRISPR-Cas9 guide RNAs, and GAPmers. Recent research has extended this approach to splice-switching oligonucleotides (SSOs), which target splicing events. Our study identifies a set of mixed-modification patterns-combining 2'-O-Methyl, 2'-MethOxyEthyl, 2'-Locked Nucleic Acid, and 2'-Constrained Ethyl ribose moieties (2'OMe, 2'MOE, LNA, and cET)-that enhance SSO potency. We term this strategy lateral mixed positional configuration, which improves SSO efficacy across various sequences and could reduce the trial-and-error process in SSO development. This advancement is supported by NAT Unlabeled Reporter Assay (NATURA), a novel platform for high-throughput quantification of NATs' functional delivery and potency. NATURA uses a reporter gene system and a comprehensive sequence library to test modifications and delivery methods, validated in a transgenic mouse model. This approach aims to accelerate NAT development and address challenges in delivering these therapies to patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing and debilitating neurodegenerative disease, yet the mechanisms underlying disease onset and progression remain poorly understood, particularly in sporadic ALS. Emerging evidence suggests that mitochondrial dysfunction and metabolic dysregulation are central to ALS pathophysiology. A key feature of ALS motor neurons (MNs) is hyper-acetylation of mitochondrial proteins, which disrupt mitochondrial respiration and energy homeostasis. In this study, we identify BLOC1S1 (also known as GCN5L1) as a novel regulator of mitochondrial acetylation in ALS. We demonstrate that BLOC1S1 is significantly upregulated in ALS patient-derived MNs, postmortem motor cortices, and spinal cords of ALS mouse models. Functional studies in induced pluripotent stem cell-derived MNs reveal that BLOC1S1 depletion rescues key disease phenotypes. Therefore, we develop an efficacious splice-switching antisense oligonucleotide that induces nonsense-mediated decay of BLOC1S1 transcripts as a potential therapeutic candidate. Besides mitigating ALS-relevant cellular deficits in MN cultures from diverse genetic backgrounds, it was validated to extend disease-free and overall survival that is associated with improved rotarod performance in an ALS mouse model. These findings establish BLOC1S1 as a critical modifier of disease progression in ALS and highlight its potential as a novel therapeutic target.

BACKGROUNDS & AIMS:Citrin deficiency (CD) is an autosomal recessive urea cycle disorder caused by biallelic loss-of-function variants in the SLC25A13 gene, leading to life-threatening hyperammonemia and hypoglycemia. Variants in deep introns can cause genetic diseases by altering splicing and are often missed by current diagnostic tools. Splice-switching oligonucleotides (SSOs) can resolve certain intronic variants, but patients harboring such variants need to be identified. We present a lean workflow from molecular diagnostics to SSO development to resolve splice-altering variants in deep introns that is applicable to other genetic disorders. METHODS:A deep intronic-gene panel was designed to identify deep intronic variants. SSOs were then developed and validated in vitro using a minigene assay and induced hepatocytes, and target engagement was verified in vivo by hydrodynamic tail vein injection of minigenes and SSOs. RESULTS:With the deep intronic-gene panel and RNA analysis, we identified a novel SLC25A13 c.469-2922G>T variant that promotes the inclusion of a premature stop codon-containing pseudo-exon, SLC25A13-PE5, thereby causing CD. Using a stepwise rational SSO design approach, we identified potent candidates inhibiting SLC25A13-PE5 at EC50 <2 nM in vitro. Upon conjugating the SSOs with GalNAc (N-acetylgalactosamine), they were validated to rescue normal protein expression and restore ureagenesis and ammonia clearance, key urea cycle functions, in patient-derived induced hepatocytes. In vivo on-target efficacy of the clinical GalNAc-SSO candidate, in the absence of acute toxicity and inflammation, was observed in a mouse model with exogenous hepatic minigene expression. CONCLUSIONS:Our data validates a platform to redefine the molecular diagnosis of urea cycle disorders and provides proof-of-concept for a precision therapy for patients with CD, for whom the only effective treatment is liver transplantation. IMPACT AND IMPLICATIONS:Deep intronic variants are common causes of genetic diseases that are commonly neglected. In this study, we demonstrate an integrated precision diagnostic and therapeutic approach for urea cycle disorders. Specifically, we focus on citrin deficiency, going from the discovery of a novel splice variant in the SLC25A13 gene with our novel deep intronic-gene panel for urea cycle disorders, to the development and in vivo validation of an efficacious splice-switching oligonucleotide candidate for the pathogenic splice variant. We envision the possibility of extrapolating this pipeline to the diagnosis and development of treatments for other rare genetic diseases.

A self-cleavable DNA nanogel loaded with splice-switch oligonucleotide (SSO) has been developed. Under acidic conditions (pH 5.0), cleavage of the acid-labile chemical linker and generation of the i-motif structure led to the disintegration of the DNA nanogel and efficient release of SSO in its unaltered native state.

Protein List and Enrichment Analysis of USP7-IP-MS

SRSF Family Proteins Essentiality in Tumor Cell Lines

Mutation Status of Selected Splicing Factors in T-ALL patients

RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

KEGG Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

Protein List and Enrichment Analysis of KGG-MS

Gene Ontology Analysis of Overlap Proteins from USP7-IP-MS and KGG-MS

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between CD4 T-Cells and T-ALL

Einleitung Mutiertes Ras (HRAS, NRAS oder KRAS) ist eines der wichtigsten Onkogene bei gastrointestinalen Tumoren und ein prognostisch ungünstiger Faktor. Die Ras-induzierte Tumorigenese verläuft in mehreren Schritten. Die Aktivierung von onkogenem Ras führt zunächst zur Induktion von Seneszenz und prämalignen Läsionen. Durch zusätzliche genetische Veränderungen (z.B. Inaktivierung von p16INK4A oder p53) kommt es zur Entstehung von malignen Tumoren.

Motif Analysis of Skip Exon Events in T-ALL Compared to CD3 T Cells