Splice-switching antisense oligonucleotide controlling tumor suppressor REST is a novel therapeutic medicine for neuroendocrine cancer.

RNA splicing regulation has revolutionized the treatment of challenging diseases. Neuroendocrine cancers, including small cell lung cancer (SCLC) and neuroendocrine prostate cancer (PCa), are highly aggressive, with metastatic neuroendocrine phenotypes, leading to poor patient outcomes. We investigated amido-bridged nucleic acid (AmNA)-based splice-switching oligonucleotides (SSOs) targeting RE1-silencing transcription factor (REST) splicing as a novel therapy. We designed AmNA-based SSOs to alter REST splicing. Tumor xenografts were generated by subcutaneously implanting SCLC or PCa cells into mice. SSOs or saline were intraperitoneally administered and tumor growth was monitored. Blood samples were collected from mice after SSO administration, and serum alanine aminotransferase and aspartate aminotransferase levels were measured to assess hepatotoxicity using a biochemical analyser. In vitro, REST_SSO reduced cancer cell viability. In a tumor xenograft model, it exhibited significant antitumor effects. It repressed REST-controlled RE1-harboring genes and upregulated miR-4516, an SCLC biomarker. Our findings suggest that REST_SSO suppresses tumorigenesis in neuroendocrine cancers by restoring REST function. This novel therapeutic approach holds promise for intractable neuroendocrine cancers such as SCLC and neuroendocrine PCa.

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

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 TGFß pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.

A Wnt-induced lncRNA-DGCR5 splicing switch drives tumor-promoting inflammation in esophageal squamous cell carcinoma.

Alternative splicing (AS) is a critical mechanism for the aberrant biogenesis of long non-coding RNA (lncRNA). Although the role of Wnt signaling in AS has been implicated, it remains unclear how it mediates lncRNA splicing during cancer progression. Herein, we identify that Wnt3a induces a splicing switch of lncRNA-DGCR5 to generate a short variant (DGCR5-S) that correlates with poor prognosis in esophageal squamous cell carcinoma (ESCC). Upon Wnt3a stimulation, active nuclear ß-catenin acts as a co-factor of FUS to facilitate the spliceosome assembly and the generation of DGCR5-S. DGCR5-S inhibits TTP's anti-inflammatory activity by protecting it from PP2A-mediated dephosphorylation, thus fostering tumor-promoting inflammation. Importantly, synthetic splice-switching oligonucleotides (SSOs) disrupt the splicing switch of DGCR5 and potently suppress ESCC tumor growth. These findings uncover the mechanism for Wnt signaling in lncRNA splicing and suggest that the DGCR5 splicing switch may be a targetable vulnerability in ESCC.

Splicing Modulation as a Promising Therapeutic Strategy for Lysosomal Storage Disorders: The Mucopolysaccharidoses Example.

Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.

2'-O-(N-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'-O-(N-(Aminoethyl)carbamoyl)methyl (2'-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2'-O-methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages. It is therefore particularly interesting and unprecedented that the 2'-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2'-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2'-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.

Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Aberrant Bcl-x splicing in cancer: from molecular mechanism to therapeutic modulation.

Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

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

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.

Nucleic Acid Therapy for ß-Thalassemia.

ß-thalassemia is caused by mutations in the ß-globin gene which diminishes or abolishes ß-globin chain production. This reduction causes an imbalance of the α/ß-globin chain ratio and contributes to the pathogenesis of the disease. Several approaches to reduce the imbalance of the α/ß ratio using several nucleic acid-based technologies such as RNAi, lentiviral mediated gene therapy, splice switching oligonucleotides (SSOs) and gene editing technology have been investigated extensively. These approaches aim to reduce excess free α-globin, either by reducing the α-globin chain, restoring ß-globin expression and reactivating γ-globin expression, leading a reduced disease severity, treatment necessity, treatment interval, and disease complications, thus, increasing the life quality of the patients and alleviating economic burden. Therefore, nucleic acid-based therapy might become a potential targeted therapy for ß-thalassemia.

Design and Application of a Short (16-mer) Locked Nucleic Acid Splice-Switching Oligonucleotide for Dystrophin Production in Duchenne Muscular Dystrophy Myotubes.

Splice-switching oligonucleotides (SSOs) have been used to modulate gene expression by interfering with pre-mRNA splicing with the intent to treat disease. For Duchenne muscular dystrophy, splicing modulation has been used to induce the skipping of exon 51 of the dystrophin transcript, allowing the production of a truncated but functional protein. Although oligonucleotide-based therapies are promising, the rapid degradation of oligonucleotides (ONs) by intracellular nucleases has been a major obstacle. Locked nucleic acid (LNA) substitution in SSOs protects oligonucleotides from nuclease degradation and enhances the hybridization properties of the oligo. However, the best optimum size of the oligo depends on the LNA substitution rate. Here we show that 16-mer DNA SSOs with 60% LNA substitution and full phosphorothioate (PS) linkage backbone efficiently induce exon 51 skipping in myogenic cells derived from a DMD patient, allowing expression of the dystrophin protein.

Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA.

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3' splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomycespombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.

Antisense Oligonucleotides for Splice Modulation: Assessing Splice Switching Efficacy.

Today, there are emerging numbers of oligonucleotide therapies being approved by the governmental authorities. These types of therapies present a different mode of action when compared to the traditional small molecules, acting at the RNA level instead of the protein level. In drug development, drug potency is defined by the drug affinity to the target biomolecule (target engagement), together with the ability to initiate a response at the molecular, cellular, tissue, or system level (efficacy). In oligonucleotide therapies, affinity and efficacy can be both easily evaluated by gene expression analysis. Although more advanced techniques can be used, this chapter presents a protocol to evaluate splice switching oligonucleotide efficacy that can be easily applied in a molecular biology laboratory without the need of advanced equipment. It describes all steps from sample preparation to data analysis.

Evaluation of Cell-Penetrating Peptide Delivery of Antisense Oligonucleotides for Therapeutic Efficacy in Spinal Muscular Atrophy.

Antisense oligonucleotides (ASOs) are a widely used form of gene therapy, which is translatable to multiple disorders. A major obstacle for ASO efficacy is its bioavailability for in vivo and in vitro studies. To overcome this challenge we use cell-penetrating peptides (CPPs) for systemic delivery of ASOs. One of the most advanced clinical uses of ASOs is for the treatment of spinal muscular atrophy (SMA). In this chapter, we describe the techniques used for in vitro screening and analysing in vivo biodistribution of CPP-conjugated ASOs targeting the survival motor neuron 2, SMN2, the dose-dependent modifying gene for SMA.

Rewriting the (tran)script: Application to spinal muscular atrophy.

Targeting RNA drastically expands our target space to therapeutically modulate numerous cellular processes implicated in human diseases. Of particular interest, drugging pre-mRNA splicing appears a very viable strategy; to control levels of splicing product by promoting the inclusion or exclusion of exons. After describing the concept of "splicing modulation", this chapter will cover the outstanding progress achieved in this field, by highlighting the breakthrough accomplished recently for the treatment of spinal muscular atrophy using two therapeutic modalities: splice switching oligonucleotides and small molecules. This review discusses the vital but feasible requirement for such drugs to deliver selectivity, and critical safety aspects are highlighted. Transformational medicines such as those developed to treat SMA are likely just the beginning of this story.

Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes.

Telomerase activity is regulated at the mRNA level by alternative splicing (AS) of its catalytic subunit hTERT. The aim of this study was to define the ability of splice-switching oligonucleotides (SSOs) that pair with hTERT pre-mRNA to induce AS and inhibit telomerase activity in human CD4+ T lymphocytes. SSOs that blocked the binding of a single splicing regulatory protein, SRp20 or SRp40, to its site within intron 8 of hTERT pre-mRNA demonstrated rather moderate capacities to induce AS and inhibit telomerase. However, a SSO that blocked the interaction of both SRp20 and SRp40 proteins with pre-mRNA was the most active. Cultivation of lymphocytes with spliced hTERT and inhibited telomerase resulted in the reduction of proliferative activity without significant induction of cell death. These results should facilitate further investigation of telomerase activity regulation, and antitelomerase SSOs could become promising agents for antiproliferative cell therapy.

Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids.

We report the evaluation of 18-mer 2'-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5' end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.

Tips to Design Effective Splice-Switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion.

Antisense-mediated exon skipping and exon inclusion have proven to be powerful tools for treating neuromuscular diseases. The approval of Exondys 51 (eteplirsen) and Spinraza (nusinersen) for the treatment of patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) was the most noteworthy accomplishment in 2016. Exon skipping uses short DNA-like molecules called antisense oligonucleotides (AONs) to correct the disrupted reading frame, allowing the production of functional quasi-dystrophin proteins, and ameliorate the progression of the disease. Exon inclusion for SMA employs an AON targeting an intronic splice silencer site to include an exon which is otherwise spliced out. Recently, these strategies have also been explored in many other genetic disorders, including dysferlin-deficient muscular dystrophy (e.g., Miyoshi myopathy; MM, limb-girdle muscular dystrophy type 2B; LGMD2B, and distal myopathy with anterior tibial onset; DMAT), laminin α2 chain (merosin)-deficient congenital muscular dystrophy (MDC1A), sarcoglycanopathy (e.g., limb-girdle muscular dystrophy type 2C; LGMD2C), and Fukuyama congenital muscular dystrophy (FCMD). A major challenge in exon skipping and exon inclusion is the difficulty in designing effective AONs. The mechanism of mRNA splicing is highly complex, and the efficacy of AONs is often unpredictable. We will discuss the design of effective AONs for exon skipping and exon inclusion in this chapter.

Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are only several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

Targeting Splicing in Prostate Cancer.

Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform specific siRNAs, and splice switching oligonucleotides.