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Development and validation of AI/ML derived splice-switching oligonucleotides

ABSTRACT: Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

SUBMITTER: Alyssa, D Fronk

PROVIDER: S-SCDT-10_1038-S44320-024-00034-9 | biostudies-other |

REPOSITORIES: biostudies-other

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Development and validation of AI/ML derived splice-switching oligonucleotides

Project description:Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFβ pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

2024-04-25 | GSE215917 | GEO

Development and validation of AI/ML derived splice-switching oligonucleotides

Project description:Development and validation of AI/ML derived splice-switching oligonucleotides

| PRJNA891287 | ENA

Development of therapeutic splice-switching oligonucleotides.

Project description:Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case.

| S-EPMC4098814 | biostudies-literature

Splice-switching antisense oligonucleotides as therapeutic drugs.

Project description:Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials.

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Anti-tumor activity of splice-switching oligonucleotides.

Project description:Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. The apoptotic regulator Bcl-x is alternatively spliced to express anti-apoptotic Bcl-x(L) and pro-apoptotic Bcl-x(S). Bcl-x(L) is upregulated in many cancers and is associated with chemoresistance, distinguishing it as an important target for cancer therapy. We previously showed that redirection of Bcl-x pre-mRNA splicing from Bcl-x(L) to -x(S) induced apoptosis in breast and prostate cancer cells. In this study, the effect of SSO-induced Bcl-x splice-switching on metastatic melanoma was assessed in cell culture and B16F10 tumor xenografts. SSOs were delivered in vivo using lipid nanoparticles. Administration of nanoparticle with Bcl-x SSO resulted in modification of Bcl-x pre-mRNA splicing in lung metastases and reduced tumor load, while nanoparticle alone or formulated with a control SSO had no effect. Our findings demonstrate in vivo anti-tumor activity of SSOs that modulate Bcl-x pre-mRNA splicing.

| S-EPMC3001088 | biostudies-other

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Project description:Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

| S-EPMC7997716 | biostudies-literature

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides.

Project description:Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)4 (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.

| S-EPMC7831936 | biostudies-literature

Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

Project description:Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

| S-EPMC4376484 | biostudies-literature

Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides.

Project description:Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15-25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

| S-EPMC6954394 | biostudies-literature

Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides.

Project description:Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon's excluded segment.

| S-EPMC8302632 | biostudies-literature

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