Alternative splicing of modulatory immune receptors in T lymphocytes: a newly identified and targetable mechanism for anticancer immunotherapy

Abstract

Alternative splicing (AS) is a mechanism that generates translational diversity within a genome. Equally important is the dynamic adaptability of the splicing machinery, which can give preference to one isoform over others encoded by a single gene. These isoform preferences change in response to the cell’s state and function. Particularly significant is the impact of physiological alternative splicing in T lymphocytes, where specific isoforms can enhance or reduce the cells’ reactivity to stimuli. This process makes splicing isoforms defining features of cell states, exemplified by CD45 splice isoforms, which characterize the transition from naïve to memory states. Two developments have accelerated the use of AS dynamics for therapeutic interventions: advancements in long-read RNA sequencing and progress in nucleic acid chemical modifications. Improved oligonucleotide stability has enabled their use in directing splicing to specific sites or modifying sequences to enhance or silence particular splicing events. This review highlights immune regulatory splicing patterns with potential significance for enhancing anticancer immunotherapy.

Introduction

Biologists have long been puzzling how the human genome, which bears considerable similarity to lower eukaryotes, is responsible for the complex, sophisticated organisms it creates. Following Sharp and Roberts’ description of RNA splicing, Gilbert, in 1978, hypothesized that alternative splicing (AS) might be the missing layer that leads to the immense protein diversity despite the only 23000-gene human genome.

RNA splicing is a “cut and paste” process, removing introns and rejoining exons from the primary gene transcript, the pre-mRNA. The process relies on the biochemical uniqueness of RNA, which DNA lacks, of extensive flexibility and intrinsic catalytic activity. Small nuclear RNAs that assemble sequentially are directed to conserved sequences in the 5’(GT) and 3’(AG) splice sites on the primary transcript in an orderly manner. Together, the small RNAs and numerous proteins form the spliceosome. An adenosine in the intronic segment performs a nucleophilic attack on the 5′ end, cleaving the 5′ nucleotide (generally the “G” in a GT); a loop is then formed and removed. Following, the exon upstream of the removed intron is ligated to the 5′ end catalyzed by the spliceosomal RNAs and the ribonuclear proteins (RNPs).

Alternative splicing produces variants that differ from the constitutive RNA transcript. It occurs parallel to the transcription process and produces several isoforms from one gene. Each isoform may lack an exon or part of an exon from either side of the constitutive exon. This pattern, called ‘cassette-type alternative exon’ or ‘exon skipping’, is the most common. Intron retention, uncommon in humans, occurs mainly in untranslated regions. See Figure 1 for common splicing patterns.

Figure 1

AS involves 95% of the genes (1). With deep RNA sequencing becoming a more common read-out in experimental systems and longer RNA reads being produced, it is now clear that the pattern of RNA splicing is dynamically regulated and constantly changes (2).

The ratio between a constitutive transcript and its alternatively spliced isoforms depends on splice site recognition, its occupancy by spliceosomal RNPs, and regulatory RNA binding proteins (RBPs) (3). These RBPs bind or complement cis-sequences on the premature transcript on the intron or the exon. Sequences that promote spliceosome assembly at a splice site are called enhancers, and their RBPs are usually serine arginine-rich proteins. Sequences that reduce splice site recognition (silencers) attract heterologous nuclear RNPs (hnRNPs) (4). It is thought that enhancers usually act to generate constitutive mRNA, while the silencers yield AS isoforms. However, how the basic mechanism of AS varies from cell to cell and what determines it remains to be further elucidated.

Here, we focus on the role of AS in the T-cell immune response, particularly on anticancer immunity. This review aims to draw attention to new therapeutic opportunities in the functional distinctions between a constitutive protein/receptor and its splice isoforms. The motivation to unveil the intriguing mechanisms by which AS amplifies and regulates immune functions relies on reports from our group and others that RNA transcripts of the same immune gene can act in different directions or magnitudes in the immune context (5, 6). Thus, AS is an essential layer of immune regulation and a potential therapeutic target.

Alternative splicing is a mechanism of dynamic adaptability

Splicing event regulation

Although the prime outcome of AS is the fold increase in functionally distinct proteins compared to the number of genes, AS also plays a significant role in the most fundamental biological processes: evolution, differentiation, and adaptation. AS is a source of evolutionary development, a determinant of organ, tissue, and cell characteristics, and part of cellular adaptation to a changing environment (2).

The concerted manner by which protein production is shifted from one isoform to another yields the regulatory characteristics of AS. Its preferences differ among tissues and developmental states and respond to extracellular signals in a dynamic manner that precedes or synchronizes with gene transcription. In parallel to the dependency of intracellular processes on transcriptional activation, cellular events emerge from the shift in protein isoform ratios. How splicing events are concerted and what network cascades occur is a field of active research emphasizing health disorders and malignancies (7). The regulation of splicing events depends on both cis-acting regulatory sequences, located in introns or exons, and trans-acting splicing factor proteins that can strengthen or weaken the spliceosome’s recognition of the splice sites (8). These regulatory proteins belong to families of RNA-binding proteins, such as arginine–serine-rich (SR), heterogeneous nuclear ribonucleoprotein (hnRNP), and RNA-binding motif (RBM) proteins (9). They recognize specific regulatory sequences and enhance or inhibit the recognition of neighboring splice sites by the core splicing machinery (7). The expression level of the regulatory proteins is tissue- and state-specific (10), and they are subjected to regulatory splicing themselves (11).

From the evolutionary point of view, alternative splicing varies significantly among species. The insertion of multiple introns that separate exons has derived from ancestral genes and predated AS in eukaryote development. The option to skip exons was enabled by DNA mutations that may have resulted in splice sites with weaker binding affinity for spliceosomal components such as U1 small nuclear ribonucleoprotein (snRNP) (12). While the emergence of alternative splice sites contributed to protein diversity and is partly unique to a species, particularly when changing the reading frame, comparative genomics indicates that sequences that regulate RNA binding proteins are conserved and shared (13, 14).

Over 200,000 identified isoforms are reported in genome databases, and the majority of them lack functional annotation. Some well-studied examples show how events of retention or exclusion of specific domains may change protein cell localization, membrane anchorage, shedding of ectodomains, mRNA stability, and translational efficiency. The molecular alterations that emerge from AS may occur without any change in the level of the general gene’s transcript or before a change (15). Furthermore, the translational changes in reading frames may produce diverse translation outputs (13) and even insert poison exons, resulting in nonsense-mediated mRNA decay (NMD) and diminished protein levels (16). We can conclude that alternative splicing is timed and regulated in a manner that is not necessarily dependent on active simultaneous gene transcription.

Alternative splicing in T lymphocytes

T-cell states are associated with preferential expression of specific splicing isoforms. A unique characteristic of T lymphocytes is that they transform within minutes from a stationary naïve or inactive state to intense activity. In their effector state, T cells must adapt to synthesize large amounts of cytokines, migrate, proliferate, lyse target cells, and address accelerated metabolic needs. Already in 2006, it was found that memory T-cells respond to antigenic stimuli faster than naïve cells by omitting exons 4, 5, and 6 from the extracellular part of the membrane phosphatase CD45. CD45 is expressed in T and B cells and, in its constitutive, full-exon inclusive state, is referred to as CD45RA. The CD45RO variant shows variable exclusion of exons 4, 5, and 6. CD45 dephosphorylates both inhibitory and costimulatory tyrosines of the Src-family kinases (17). Oberdoerffer et al. showed that the transition from the RA to the RO form depends on the activity of the splicing factor hnRNPLL (heterogeneous ribonucleoprotein L-like) (18). HnRNAPLL was suggested to be a master regulator in activated T cells, affecting not only CD45.

Before and in parallel to CD45, specific gene isoforms impacting T-cell function were discovered. Interesting events recorded in activated T-cells included the short isoform of CD28, which induces faster activation (19), splice variants of CD44 and CTLA4, which correlated with a higher risk of autoimmune disease (20, 21), and MALT1A, a paraprotease that integrates TCR activation with the downstream IKK/NF-κB pathway. Reminiscent of CD45, naïve T-cells express MALT1B, a splice isoform missing exon 7, while activated T-cells express MALT1A, which includes exon 7 and is associated with rapid NF-κB signaling and improved lymphocyte function (22, 23).

A landscape view of AS in immune cells was offered by Lynch et al. in 2004, preceding a complete landscape of the comprehensive gene involvement in this phenomenon (22). Although the list of spliced genes described to regulate lymphocyte activation was restricted, the diverse array of functions governed by splicing suggested the substantial ubiquity of this process (23).

In the last few years, analyses have focused on gene families and activation cascades as it becomes clear that AS affects most genes. An example is the production of anti-apoptotic splice isoforms of members of the BCL2 gene family in activated T-cells. Adding costimulation via CD28 increased the ratio of anti-apoptotic splice variants and augmented T-cell proliferation. Interestingly, the genes that displayed significant changes in their splice isoform ratios did not have the highest expression levels (24).

The concept of AS-induced effector transition is not limited to activated T lymphocytes but also plays a critical role in B cell affinity maturation. In these processes, poly-pyrimidine tract binding proteins PTBP1 and PTB3 are splicing factors that drive the appropriate expression of gene sets required to adapt B lymphocytes to antibody-producing cells (25).

Splicing events that generate soluble isoforms of immune receptors

A prevalent splicing pattern observed in immune receptors gives rise to soluble isoforms that lack membrane anchorage and are secreted into the extracellular space. These soluble receptors may regulate signaling cascades, which differ from those initiated by their parental receptor (Table 1). Most prominently, the soluble receptors can function as a decoy of their corresponding ligands and compete with their constitutive, membrane-bound forms (26–35). The ratio between the membranal and the soluble isoforms of a receptor can remain fixed (26, 36). However, it might change depending on the cell’s metabolic, functional, or differentiation state (37, 38). Diverting the pre-mRNA splicing towards the soluble isoforms results in reduced expression of the membrane-bound receptor and can even negate its cellular effect. In a different context, the soluble receptors may have agonistic effects (28, 39) and initiate reverse signaling by binding to other receptors (40–42). For example, glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand that is expressed on plasmacytoid dendritic cells prompts a reverse signal that initiates noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase upon binding to soluble GITR. This leads to the tryptophan catabolism immunoregulatory pathway (43). In addition, soluble isoforms have been documented to bind with their ligand to distinct membranal partners, activating trans-signaling pathways (44, 45). Furthermore, some soluble receptors stabilize their ligand configuration or alter their biodistribution (31, 45–47). Another typical example of important alternative splicing of immune cells is the removal of the hydrophobic transmembranal segment of the B-cell receptor to form a secreted immunoglobulin (48). Interestingly, soluble receptors may exert different functions (i.e., agonistic and antagonistic) depending on their concentration (45, 46, 49) (Figure 2).

Figure 2

The common splicing patterns that lead to the generation of soluble isoforms of membranal receptors include (1) Transmembrane domain (TMD) skipping: In the process of alternative splicing, skipping of the transmembrane encoding exon results in the creation of a soluble product encompassing both the intracellular and extracellular domains (26, 33, 50–55); (2) Alternative terminator: a shortened soluble isoform is encoded by a sequence that includes a mutually exclusive exon containing an alternative polyadenylation site, or by alternative splicing that results in frameshift and premature stop codon (48, 56, 57). As a result, the translated proteins include only the extracellular domain, lose their membrane anchorage and become soluble (27, 58, 59).

It should be noted that alternative splicing is not the sole mechanism that creates soluble receptors. These segments can also be made by proteolytic cleavage of extracellular domains by proteases in the extracellular matrix (Figure 3). However, unlike AS, the intracellular domain (ICD) of a cleaved receptor remains anchored and theoretically may retain its effect. The impact of a truncated signaling domain is diverse or unknown. Typical examples of receptors that utilize both mechanisms to produce their soluble formats are cytokine receptors, including the TNF and TNFR superfamily (59, 60). In addition, some immune receptor genes lack the transmembrane domain and are, therefore, constitutively expressed as soluble receptors with linked intracellular and extracellular domains. They mainly function as decoy receptors (61). For example, decoy receptor 3 (DcR3, TNFRSF6B) is a secreted TNFR superfamily member that lacks a transmembrane domain. DcR3 can interrupt FAS-FASL interaction by binding FASL and inhibiting FASL-induced apoptosis (62).

Figure 3

Alternative splicing of the immunoglobulin superfamily

The immunoglobulin (Ig) superfamily is a large group of proteins with a common Ig domain. The Ig superfamily is critical in the immune response networks (63). Some Ig superfamily receptors can be translated to a soluble form by an alternative splicing process (64, 65). Among these are CTLA4 (39, 58), CD83 (42, 66), and LAG3 (66). Here, we will focus on two specific examples: The programmed cell death receptor PD-1 and the B cell receptors (BCRs) that convert, after splicing, into immunoglobulins (antibodies).

PD-1

Following stimulation, PD-1 is expressed on T-cells in a membrane-bound form (mPD-1). When it binds to its ligand (PD-L1), mPD-1 inhibits the effector functions of T- cells, promotes apoptosis, and restricts proliferation (67, 68). PD-1 exon 3, which encodes the transmembrane domain, can be skipped by alternative splicing, generating a soluble receptor form (sPD-1). The ratio between the two isoforms is consistent during T-cell activation (36). sPD-1 can act as a decoy receptor and compete with the PD-1 receptor on the interaction with the ligands PD-L1 and PD-L2, and block the interaction of PD-L1 with B7-1 (69). The shedded PD-1 ectodomain exerts a similar effect, suppressing the PD-1 inhibitory function (70, 71). It has been speculated that sPD-1 has a reverse signaling effect when binding to PD-L1 on dendritic cells (41).

Immunoglobulins can be membrane-bound or secreted as antibodies. Naive B cells express membrane-bound receptors, usually from the IgM class. Following stimulation, the B cell receptors undergo alternative splicing via an alternative terminator mechanism. As a result, the carboxy terminus no longer contains the hydrophobic transmembrane domain but, instead, has a hydrophilic secretory tail. The secreted antibodies play a crucial independent role during the immune response (48).

Alternative splicing of the TNF-receptor superfamily

The TNFRSF comprises trimeric receptors made of three homologous molecules that initiate signaling pathways involved in inflammation, proliferation, differentiation, cell migration, and induction of cell death (72). Like the Ig superfamily, TNFRSF members share similar splicing patterns that result in soluble isoforms.

FAS (CD95, TNFRSF6) is one of the best-known members of the TNF receptors superfamily. It is abundantly expressed in many tissues, including the gastrointestinal tract, the respiratory system, and lymphoid tissues (73–75). FAS is mainly known for its pro-apoptotic pathway activation following FAS ligand (FASL) binding (76). However, it also has other functions, e.g., it takes part in the differentiation of naïve T cells to memory cells (77). FAS is robustly expressed on T-cells and has an apoptosis-inducing role during T-cell development (78) and activation (79–81). A specific alternative splicing event is the skipping of exon 6, which encodes the transmembrane domain of FAS, resulting in a soluble form of the FAS receptor (52, 53). The FAS exon 6 skipping mechanism has been studied extensively. It has been shown that many splicing factors can regulate this event, among them TIA-1 (82), PTB (82), HuR (83), hnRNP A1 (84), SRSF4 (85), SRSF7 (86), and SRSF6 (87). Similarly to PD-1, the soluble FAS receptor competes with membrane-bound FAS for FASL ligation, thereby limiting FAS signaling (30, 52). Bajgain et al. described the ability of secreted FAS extracellular domain to enhance CAR T-cell antitumor activity against a FAS-ligand-expressing tumor (88).

TGFβ (transforming growth factor beta receptor) TGFβ is of special interest because it controls immunity via a rich network of cells and mediators, with the end result being immune evasion of the cancer tissue. The biological functions of TGFβ are mostly mediated by the monomeric, soluble form of the protein. The monomer is cleaved by proteases in the Golgi complex and later released from glycoproteins that ligate it in a non-covalent manner (89). TGFβ enhances the expansion of regulatory T cells (Tregs), the inhibition of NK and effector T cells, and the induction of immune suppressive cytokines including IL-4 and IL-10. Active TGFβ exerts its effect via receptors that activate SMAD transcription factors, a family with hundreds of regulatory elements. Tumors exploit TGFβ to induce a supportive stroma that weakens the immune response by acting as a mechanical barrier and expressing inhibitory membranal ligands, such as PD-L1 (90, 91). In a series of patients with gynecological cancers who received immune checkpoint inhibitors, a high TGFβ expression score correlated with treatment failure and reduced survival (92). The type II receptor for TGFβ has a splicing variant which lacks the transmembrane domain, and exert a higher binding affinity to the three sub-types of TGFβ. By doing so, it competes with the natural ligands and reduces fibrotic pathology (93).

Type I cytokine receptors

In addition to the Ig and TNFR superfamilies, members of other immune receptor families can generate soluble forms through alternative splicing, including type I cytokine receptors IL6Rα (54, 94), IL-4Rα (57), and IL-7Rα (33). Another example is the common γ chain IL-2, 7, and 15 cytokine receptors, for which skipping exon 6 encoding the transmembrane domain results in a frameshift and a premature stop codon. The resultant proteins contain only the extracellular domain. The soluble IL-2 and IL-7 receptors were reported to impair T-cell signaling and function (27).

Pathology of splicing and alternative splicing

Splicing is an imperative regulator of most cellular functions. Therefore, disrupted splicing regulation can lead to different pathologies, depending on the involved tissue and the protein products of the aberrant transcript. The most investigated pathologies that result from erroneous splicing events include neurodegenerative disorders and cancers. The first arise from germline mutations, while the latter arise from somatic genome aberrations. However, splicing-related mutations can cause many other disorders, such as dilated cardiomyopathy and Marfan syndrome (109, 110).

It remains a mystery why germline splicing-related mutations primarily affect the brain. One theory holds that alternative splicing is crucial in determining the neural cell state (19) and that neural tissue is rich in tissue-specific splicing events. However, not all splicing-related mutations in neural cells lead to a change in alternative splicing. An example of this is Duchenne muscular dystrophy (DMD), where a deletion of an exon leads to the production of a truncated protein via the process of nonsense-mediated decay (NMD) rather than a new isoform of the original protein (111).

Some argue that 15-50% of pathological mutations affect gene splicing (9–11). Nevertheless, these diseases are not regarded as splicing-related disorders since mutations that do not change the coding sequence are typically misclassified as allelic variations (112–114). In addition, the wide use of exome sequencing, which filters out most intronic parts, introduces an inherent bias underscoring splicing mutations (115–118).

Dis-regulated splicing leading to neurodegenerative disorders

Neurodegenerative diseases are a group of disorders caused by the gradual loss of neuronal cell function or structure. Strikingly, splicing-related mutations are one of the leading causes of many neurodegenerative diseases (119). The most investigated neuronal disorder instigated by splicing is spinal muscular atrophy (SMA). Nonetheless, most neural pathologies, such as early-onset Parkinson’s (119–121), Alzheimer’s disease (122), familial dysautonomia (123), and Amyotrophic Lateral Sclerosis (ALS), could evolve from splicing mutations (124, 125).

Given the significant number of neurodegenerative disorders caused by mutations impacting RNA splicing, it is not surprising that there have been numerous efforts to investigate the use of splicing-editing techniques as a treatment option for these conditions. For example, antisense oligonucleotides (ASOs) are being widely researched for their potential use in treating SMA, DMD, and ALS (126). The use of ASOs to manipulate alternative splicing is further discussed elsewhere in this review. The use of CRISPR-Cas9 to affect alternative splicing has been suggested for Duchenne muscular dystrophy (127), and spliceosome-mediated RNA trans-splicing (SMaRT) (128) has been tested in Huntington’s disease (129), DMD (130), and Alzheimer’s disease (131).

Dis-regulated splicing causing cancer and immune evasion

Splicing-related mutations in cancer can be grouped into three categories: 1-those affecting the core spliceosome complex, resulting in new isoforms; 2-those impacting splicing factors, affecting the expression levels of multiple isoforms; and 3-those affecting splicing recognition sites, altering the expression level of a single gene or creating new isoforms (Table 2; Figure 4).

Figure 4

Splicing factors mutations are particularly prevalent in myeloid neoplasms; for example, SF3B1, that increases anti-apoptotic isoforms, enhances tumor proliferation and progression, and is associated with poor survival of patients (134, 140, 141, 180, 181). U2AF1 is another splicing factor mutated in myeloid malignancies that drives altered splicing preferences. Intronic mutations are more frequent than exonic, and a third of somatic mutations in the exon-intron boundary are associated with splicing changes. If a mutation occurs in a 5’ or 3’ splicing site, there is a greater than 50% chance of it leading to a splicing shift (182).

Splicing can be employed as a cancer treatment approach in various forms: using single-stranded oligonucleotides to change the splicing of specific genes and switch between oncogenic and tumor-suppressing forms, as has been demonstrated for the BCL gene (67); regulating specific splicing factors through drugs that directly impact them, such as blocking SF3B1 (68); or by attacking the pathway which the mutant splicing factor exploits. Thus, tumors with driver mutations in SF3B1 or U2AF1 may be vulnerable to NMD inhibition (68–72). Some widely used therapies, such as camptothecin and cisplatin, have been found to impact RNA splicing, potentially contributing to their efficacy (73–75).

As discussed in the following paragraph, recent attention has focused on the generation of neo-antigens by including erroneous transcripts. However, altered splicing and the emergence of usually unexpressed isoforms independently impact tumor immunogenicity. These effects often hinder the anticancer immune response. For example, HLA tumor-enriched alternative splicing events occur in 10-30% of lung and breast cancers, affecting MHC expression. When HLA expression is inconsistent, the ability of tumor epitopes to be presented and recognized is diminished or completely lost (183). In ovarian cancer, certain splicing factors, such as BUD31, SF3B4, and CTNNBL1, may indirectly support immune evasion (184). This immune escape may involve increased PD-L1 expression and primary resistance to PD-1 inhibitors. Such mechanisms are seen in clear renal cell carcinoma, where an exon-including splicing event in the chromatin remodeling gene PBRM1 contributes to immune evasion (185).

Generation of cancer neo-antigens by mutations in splicing factors

While reports indicate that altered splicing isoforms contribute to tumor immune evasion, splicing alterations are now attracting significant interest as a source of cancer antigenicity. This interest stems from the potential of AS to drive isoforms that include retained intronic sequences. These intronic transcripts, in turn, may form neoantigens—peptide sequences that have not had the opportunity to tolerize the immune system. Such newly transcribed sequences hold the potential for generating protective immunity and improving clinical responses to immune checkpoint inhibitors (61–64).

Several pharmacological compounds have been used that either degrade splicing factors, disrupt spliceosome assembly, or inhibit nonsense-mediated decay (186, 187). One example is indisulam, an anticancer sulfonamide that generates aberrant transcripts. Interestingly, indisulam does not directly inhibit cancer growth; instead, it triggers a T-cell response against cryptic sequences from abnormal RNA, which impedes tumor progression. Other splicing-disruptive compounds, such as pladienolide B and H3B-8800, are currently being evaluated in experimental systems and clinical trials for myeloid neoplasms. Predicting the effect of splice manipulation on the tumor microenvironment is challenging, but as will be discussed, induction of soluble ectodomains from immune-modulatory receptors may interfere with immune checkpoint inhibitors. Soluble PD1, for instance, may saturate PD-1 blocking antibodies and reduce their availability to rescue exhausted antitumor T cells (188).

RNA sequencing for splicing analysis

The technology developed to sequence RNA and obtain long transcript reads that capture added or missing nucleotides was crucial to assessing AS in health and disease.

Bulk RNA sequencing (RNA-seq) is mainly performed using two methodologies (Figure 5). The first is short-read sequencing, which can sequence RNA molecules in reads of up to 301 base pairs (bp), for which the Illumina platform is commonly used. The second is long-read sequencing, known as “third-generation sequencing.” This method can sequence up to 26,000 bp RNA molecules in the NanoporeTM platform (189). Long-read sequencing has the advantage of identifying full-length transcripts derived from each gene. However, this sequencing method had an accuracy of only 90% and is, therefore, error-prone. Erroneous sequencing interferes with the alignment of the reads to a reference genome and thus can miss sutured exons and their splice junction, an important feature required to determine the splicing pattern (190). However, recently, Nanopore announced that its sequencing accuracy has increased to 99.9%.

Figure 5

Since Illumina sequencing is well-established and widely used, most splicing analysis tools are designed for short reads. Analyzing bulk RNA-seq from Illumina data can be done in three ways. The first is determining the exon expression level and comparing its expression in varying biological settings or states. This method is called “exon-based”. The second method aims to deduce isoform expression from the short reads sequencing. This method is called “isoform-based”. The third approach, called “event-based,” computes the relative inclusion of an exon between two exons. This approach utilizes reads of splice junctions that overlap at least two exons.

A comparison of the main computational tools based on these three methods concluded that the event-based and exon-based tools while having a relatively low overlap, seem to work the best. It is suggested that concurrent use of the two methods yields the optimal splicing map of a given cellular population (191).

Another critical parameter to consider when performing splicing analysis is the quality of the RNA-seq data. In this regard, two features need to be accounted for: the depth of the sequencing and the length of the reads. Mehmood et al. (191) have noted that a depth between 40-60 million reads per sample will be sufficient for a robust splicing analysis. When considering reading length, 100 bp reads were the threshold for thoroughly detecting splicing junctions (192). It is also advised to sequence the data using paired-end sequencing to increase the read length.

Extracting splicing data from single-cell RNA-seq is even more complex. In general, to apply splicing analysis tools, the samples must be produced to capture the full transcript. However, most single-cell RNA-seq technologies are based on a 3’ or 5’ capturing of the RNA molecule. As a result, while preparing the sequencing libraries, only the transcript’s end is included; thus, there are limited options for splicing analysis (193). The main exception to these technologies is Smart-seq sequencing, which captures reads from all over the transcript. This technology enables splicing analysis with the limitation of read depth and length. This was demonstrated with the single-cell splicing analysis tool ‘Expedition.’ In their study, Yan Song et al. (194) used Smart-seq2 sequencing with a mean of 25 million reads per cell and a 100 bp read length. In comparison, 10XGenomics™ recommends a sequencing depth of 20,000-50,000 reads per cell and a read length of 28 bp (195); this is shallow sequencing compared to Yan Song’s analysis. To overcome these problems, a new single-cell long-read RNA sequencing technology based on Pacific Bioscience’s sequencing, called MAS-ISO-seq, was recently introduced. This technology is still new and needs further investigation. Furthermore, a joint project of Nanopore and 10x Genomics produced long-reads in single-cell RNA sequencing (196).

Splicing modification using antisense oligonucleotides

During the 70s, evidence accumulated for the promising ability of small RNA molecules to control translation processes (197–199). Paterson et al. were the first to generate a translation-inhibiting system based on a complementary mRNA-DNA hybrid, resulting in reversibly arrested β globin translation (199). In 1978, Paul Zamecnik and Mary Stephenson used synthetic DNA against RSV (200). Their 13-nucleotide product hybridized with the viral mRNA and prevented viral replication (201). Later, it was shown that the mechanism of action of the short, single-stranded oligonucleotides included RNase-H1 assembly to the DNA-mRNA hybrid, cleavage, and mRNA degradation (202). Despite the promising results, the research in this field was paused for a decade, mainly due to technical issues related to nucleotide synthesis (203, 204), skepticism about the ability of nucleic acids to enter target cells, and restricted knowledge of the human genome (205). The progress in these aspects re-ignited the research in nucleic acids-based manipulation. The chemically modified, short, single-stranded antisense oligonucleotides (ASOs) improved durability, cellular uptake, delivery, and post-transcriptional effects.

Chemical modifications

The advancement of chemical modifications of nucleic acids marked a significant milestone in the clinical application of this compound class. A key outcome was the development of splice-switching antisense oligonucleotides (SSOs), designed to modify alternative splicing patterns and enhance exon skipping. Specifically, chemical modifications that reduce RNaseH activity form a stable DNA-mRNA hybrid, preventing subsequent RNA degradation (206). These SSOs can be directed towards splice-site sequences, hindering and redirecting the spliceosome to an alternative splice site in the subsequent exon (207, 208) (Figure 6).

Figure 6

In addition to splicing alterations via complementation to splice sites, SSO can modify splicing by targeting splicing enhancers (ESE, ISE) or silencers (ESS, ISS) (209, 210). These interventions may interrupt splicing by inhibiting linkage to splicing factors, leading to exon exclusion or inclusion. In addition to whole exon skipping, the pre-mRNA splicing modulation can result in intron retention, alternative 5′ and 3′ splice sites, alternative promoter, or alternative polyadenylation sites (209).

Finding SSO-targetable splicing motifs is not trivial. A systematic scan of the exon of interest is necessary to spot the precise sequence, which the SSO should complement to alter the wild-type splicing pattern.

Two types of chemical modification are currently used for FDA-approved drugs: 2′-O-methoxyethyl (2′-MOE) nucleosides with phosphorothioate (PS) backbone and phosphorodiamidate morpholino oligomers (PMO) with a N, N-dimethylamino phosphorodiamidate backbone (Figure 7).

Figure 7

2’-MOE belongs to a group of modifications in the 2’O of the furanose ring of the nucleic acid. Other prevalent modifications are 2′-O-methyl (2′-OMe), locked nucleic acid (LNA), and SSOs containing 2′-constrained ethyl (2′-cEt). Alongside the RNaseH1 resistance, the 2’O modifications increase the SSO affinity (211). High affinity is attributed to higher potency, longer half-life, and less immune-provoking properties (212, 213). 2’MOE modifications are usually accompanied by switching Oxygen in the backbone to Sulfur (PS). This switch decreases the SSO affinity but improves the resistance to nuclease activity (214, 215) and molecular binding to proteins – resulting in reduced kidney clearing (216) and improved uptake by target cells (217–219).

In PMO (220), a morpholino ring replaces the furanose ring. In addition, the negatively charged backbone is replaced by a N, N-dimethylamino phosphorodiamidate backbone. As a result of these changes, the SSOs have higher in vivo tolerance but faster kidney clearance, which requires a higher dosage (221, 222).

Although these are the main modifications currently used for SSO drugs, recent publications have shown how additional chemical modifications can further improve splicing modulation. For example, Langner et al. synthesized a hybrid that combines PMO modification with a PS backbone, which exhibits higher efficiency than 2’-MOE modification with the same backbone (223).

SSO-based drugs and clinical trials

Eighteen RNA-targeted oligonucleotide drugs have been approved, including five SSOs (206). The most advanced examples of clinical use of SSOs are in the field of genetic neuromuscular diseases.

The first SSO that the FDA approved is used for spinal muscular atrophy (SMA) treatment. The drug nusinersen, approved by the FDA in 2016, is an SSO with a 2′-MOE modification and a PS backbone. Nusinersen targets the splicing silencer located in SMN2 intron 7 pre-mRNA, and by blocking the binding of hnRNPA1 and A2, it promotes higher exon 7 inclusion, increasing the SMN2 protein synthesis (224, 225). The treatment results in prolonged survival and a dramatic improvement in motor development.

Other approved SSO drugs are used for the treatment of Duchenne muscular dystrophy (DMD). This severe, progressive muscle-wasting disease causes difficulty in movement and breathing and, eventually, early death. It is caused by mutations in the DMD gene, leading to impaired dystrophin protein production (226). In recent years, the FDA has approved four drugs based on a mechanism of SSO with PMO modification. The first drug approved, eteplirsen, was approved in 2016 and causes mutated exon 51 skipping (227). Three additional drugs that work in a similar mechanism have been approved in recent years for different mutations that lead to DMD: golodirsen, which causes exon 53 skipping, was approved in 2019 (228); viltolarsen, approved in 2020, also causes exon 53 skipping (229); and casimersen, approved in 2021, induces exon 45 skipping (230). To date, DMD is the only disease for which even modest, consistent clinical benefit has been shown using PMOs. Thus, PMO SSOs have demonstrated minimal and doubtful applicability in mammalian systems (227, 231).

Considering the achievements of SSOs in DMD and SMA, several groups have recently published promising data demonstrating the potential of ASO in other diseases. For example, Yang et al. (232) show the use of SSO to prevent a splicing pattern that arises from an alternative 3’ splice site between SYNGAP1 exon 10 and exon 11. This splicing pattern leads to nonsense-mediated decay (NMD). Mutations in this gene are a common cause of autism and intellectual disability. Using SSO with 2′-MOE modification increased the expression of the active protein in an in vitro system. Promising results for the use of SSO can also be seen in the treatment of Dravet syndrome (233), Huntington’s disease (234), and fragile X syndrome (235). Similarly, in cystic fibrosis, Oren et al. (236) and Michaels et al. (237) demonstrate the use of SSO that leads to mutated exon 23 skipping, increasing the expression of the CFTR protein.

SSO and cancer treatment

The use of SSO in cancer treatment is still in its early stages. There is currently no approved drug, but there are ongoing research studies. The primary approach for anticancer SSO is modulating the alternative splicing of oncogenes toward NMD, nonfunctional dominant negative isoforms, or isoforms with the opposite function.

For example, Dewaele et al. (238) used PMO-modified SSO for MDM4 exon 6 skipping, resulting in nonsense-mediated decay and rescue of MDM4’s target - the tumor suppressor protein p53. The SSO administration reduces diffuse large B cell lymphoma growth both in vitro and in vivo.

Using SSOs for translatable alternative splicing isoforms was shown in the human epidermal growth factor receptor 2 (HER2) case. HER2 is an oncogene and established therapeutic target in a large subset of women with breast cancer (239). Wan et al. (240) and Pankratova et al. (241) used SSOs to skip HER2 exons 15 and 19, respectively. The manipulations resulted in the upregulation of Δ15HER2, a HER2 inhibitor isoform, and Δ19HER2, a dominant negative isoform, leading to apoptosis and inhibition of proliferation.

Khurshid et al. (242) recently proposed using SSO for patients with rhabdomyosarcoma (RMS). In their article, the group describes the modification of the insulin receptor splicing pattern by targeting the binding site of the splicing factor CELF1. This prevents the skipping of exon 11, leading to an increase in the expression of the receptor in its full form (IR-B). The use of SSO in an RMS cell line system led to a decrease in proliferation, migration, and angiogenesis.

Manipulating cancer-associated metabolic programs using SSO was demonstrated by Wang et al. (243). The group found that elements in exon 10 of the pyruvate kinase M (PKM) gene influence the choice between the inclusion of exon 10 and exon 9. Exon 10 inclusion, the M2 isoform, is common in cancer tumors and is associated with their ability to switch to aerobic glycolysis (Warburg effect). The group demonstrated the possibility of using SSO for splicing modulation in favor of exon 9 inclusion and showed that the manipulation could lead to apoptosis of glioblastoma cell lines. Recently, the group showed a similar effect in a hepatocellular carcinoma mouse system (244). In summary, the use of SSOs to manipulate the immune system is still in its early stages. While there is significant progress in understanding the immune system at the molecular landscape, many complexities regarding the manipulation of T cells are yet to be unraveled.

References

• 1

  Nilsen TW Graveley BR . Expansion of the eukaryotic proteome by alternative splicing. Nature. (2010) 463:457–63. doi: 10.1038/nature08909

  • CrossRef
  • Google Scholar

• 2

  Baralle FE Giudice J . Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. (2017) 18:437–51. doi: 10.1038/nrm.2017.27

  • CrossRef
  • Google Scholar

• 3

  Barash Y Calarco JA Gao W Pan Q Wang X Shai O et al . Deciphering the splicing code. Nature. (2010) 465:53–9. doi: 10.1038/nature09000

  • CrossRef
  • Google Scholar

• 4

  Holmes ME Hertel KJ . Interdependent regulation of alternative splicing by SR and hnRNP proteins. bioRxiv. (2024). doi: 10.1101/2024.08.19.608666

  • CrossRef
  • Google Scholar

• 5

  Hajaj E Zisman E Tzaban S Merims S Cohen J Klein S et al . Alternative splicing of the inhibitory immune checkpoint receptor SLAMF6 generates a dominant positive form, boosting T-cell effector functions. Cancer Immunol Res. (2021) 9:637–50. doi: 10.1158/2326-6066.CIR-20-0800

  • CrossRef
  • Google Scholar

• 6

  Hanawa H Ma Y Mikolajczak SA Charles ML Yoshida T Yoshida R et al . A novel costimulatory signaling in human T lymphocytes by a splice variant of CD28. Blood. (2002) 99:2138–45. doi: 10.1182/blood.V99.6.2138

  • CrossRef
  • Google Scholar

• 7

  Bonnal SC Lopez-Oreja I Valcarcel J . Roles and mechanisms of alternative splicing in cancer - implications for care. Nat Rev Clin Oncol. (2020) 17:457–74. doi: 10.1038/s41571-020-0350-x

  • CrossRef
  • Google Scholar

• 8

  Lee Y Rio DC . Mechanisms and regulation of alternative pre-mRNA splicing. Annu Rev Biochem. (2015) 84:291–323. doi: 10.1146/annurev-biochem-060614-034316

  • CrossRef
  • Google Scholar

• 9

  Fu XD Ares M Jr. Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet. (2014) 15:689–701. doi: 10.1038/nrg3778

  • CrossRef
  • Google Scholar

• 10

  Badr E ElHefnawi M Heath LS . Computational identification of tissue-specific splicing regulatory elements in human genes from RNA-seq data. PloS One. (2016) 11:e0166978. doi: 10.1371/journal.pone.0166978

  • CrossRef
  • Google Scholar

• 11

  Lareau LF Brenner SE . Regulation of splicing factors by alternative splicing and NMD is conserved between kingdoms yet evolutionarily flexible. Mol Biol Evol. (2015) 32:1072–9. doi: 10.1093/molbev/msv002

  • CrossRef
  • Google Scholar

• 12

  Carmel I Tal S Vig I Ast G . Comparative analysis detects dependencies among the 5’ splice-site positions. RNA. (2004) 10:828–40. doi: 10.1261/rna.5196404

  • CrossRef
  • Google Scholar

• 13

  Magen A Ast G . The importance of being divisible by three in alternative splicing. Nucleic Acids Res. (2005) 33:5574–82. doi: 10.1093/nar/gki858

  • CrossRef
  • Google Scholar

• 14

  Brooks AN Yang L Duff MO Hansen KD Park JW Dudoit S et al . Conservation of an RNA regulatory map between Drosophila and mammals. Genome Res. (2011) 21:193–202. doi: 10.1101/gr.108662.110

  • CrossRef
  • Google Scholar

• 15

  Dillman AA Hauser DN Gibbs JR Nalls MA McCoy MK Rudenko IN et al . mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex. Nat Neurosci. (2013) 16:499–506. doi: 10.1038/nn.3332

  • CrossRef
  • Google Scholar

• 16

  de Oliveira Freitas MaChado C Schafranek M Bruggemann M Hernandez Canas MC Keller M Di Liddo A et al . Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia. Nucleic Acids Res. (2023) 51:870–90. doi: 10.1093/nar/gkac1225

  • CrossRef
  • Google Scholar

• 17

  Salmond RJ McNeill L Holmes N Alexander DR . CD4+ T cell hyper-responsiveness in CD45 transgenic mice is independent of isoform. Int Immunol. (2008) 20:819–27. doi: 10.1093/intimm/dxn040

  • CrossRef
  • Google Scholar

• 18

  Oberdoerffer S Moita LF Neems D Freitas RP Hacohen N Rao A . Regulation of CD45 alternative splicing by heterogeneous ribonucleoprotein, hnRNPLL. Science. (2008) 321:686–91. doi: 10.1126/science.1157610

  • CrossRef
  • Google Scholar

• 19

  Zhang X Chen MH Wu X Kodani A Fan J Doan R et al . Cell-type-specific alternative splicing governs cell fate in the developing cerebral cortex. Cell. (2016) 166:1147–62 e15. doi: 10.1016/j.cell.2016.07.025

  • CrossRef
  • Google Scholar

• 20

  Latini A Novelli L Ceccarelli F Barbati C Perricone C De Benedittis G et al . mRNA expression analysis confirms CD44 splicing impairment in systemic lupus erythematosus patients. Lupus. (2021) 30:1086–93. doi: 10.1177/09612033211004725

  • CrossRef
  • Google Scholar

• 21

  AlFadhli S . Overexpression and secretion of the soluble CTLA-4 splice variant in various autoimmune diseases and in cases with overlapping autoimmunity. Genet Test Mol Biomarkers. (2013) 17:336–41. doi: 10.1089/gtmb.2012.0391

  • CrossRef
  • Google Scholar

• 22

  Ruefli-Brasse AA French DM Dixit VM . Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase. Science. (2003) 302:1581–4. doi: 10.1126/science.1090769

  • CrossRef
  • Google Scholar

• 23

  Meininger I Griesbach RA Hu D Gehring T Seeholzer T Bertossi A et al . Alternative splicing of MALT1 controls signalling and activation of CD4(+) T cells. Nat Commun. (2016) 7:11292. doi: 10.1038/ncomms11292

  • CrossRef
  • Google Scholar

• 24

  Blake D Radens CM Ferretti MB Gazzara MR Lynch KW . Alternative splicing of apoptosis genes promotes human T cell survival. Elife. (2022) 11. doi: 10.7554/eLife.80953.sa2

  • CrossRef
  • Google Scholar

• 25

  Monzon-Casanova E Screen M Diaz-Munoz MD Coulson RMR Bell SE Lamers G et al . The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers. Nat Immunol. (2018) 19:267–78. doi: 10.1038/s41590-017-0035-5

  • CrossRef
  • Google Scholar

• 26

  Khan M Zhao Z Arooj S Fu Y Liao G . Soluble PD-1: predictive, prognostic, and therapeutic value for cancer immunotherapy. Front Immunol. (2020) 11:587460. doi: 10.3389/fimmu.2020.587460

  • CrossRef
  • Google Scholar

• 27

  Hong C Luckey MA Ligons DL Waickman AT Park JY Kim GY et al . Activated T cells secrete an alternatively spliced form of common gamma-chain that inhibits cytokine signaling and exacerbates inflammation. Immunity. (2014) 40:910–23. doi: 10.1016/j.immuni.2014.04.020

  • CrossRef
  • Google Scholar

• 28

  Ward FJ Dahal LN Wijesekera SK Abdul-Jawad SK Kaewarpai T Xu H et al . The soluble isoform of CTLA-4 as a regulator of T-cell responses. Eur J Immunol. (2013) 43:1274–85. doi: 10.1002/eji.201242529

  • CrossRef
  • Google Scholar

• 29

  Li X Li J Zheng Y Lee SJ Zhou J Giobbie-Hurder A et al . Granulocyte-macrophage colony-stimulating factor influence on soluble and membrane-bound ICOS in combination with immune checkpoint blockade. Cancer Immunol Res. (2023) 11:1100–13. doi: 10.1158/2326-6066.CIR-22-0702

  • CrossRef
  • Google Scholar

• 30

  Papoff G Cascino I Eramo A Starace G Lynch DH Ruberti G . An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro. J Immunol. (1996) 156:4622–30. doi: 10.4049/jimmunol.156.12.4622

  • CrossRef
  • Google Scholar

• 31

  Sato TA Widmer MB Finkelman FD Madani H Jacobs CA Grabstein KH et al . Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo. J Immunol. (1993) 150:2717–23. doi: 10.4049/jimmunol.150.7.2717

  • CrossRef
  • Google Scholar

• 32

  Smith DE Hanna R Della F Moore H Chen H Farese AM et al . The soluble form of IL-1 receptor accessory protein enhances the ability of soluble type II IL-1 receptor to inhibit IL-1 action. Immunity. (2003) 18:87–96. doi: 10.1016/S1074-7613(02)00514-9

  • CrossRef
  • Google Scholar

• 33

  Crawley AM Faucher S Angel JB . Soluble IL-7R alpha (sCD127) inhibits IL-7 activity and is increased in HIV infection. J Immunol. (2010) 184:4679–87. doi: 10.4049/jimmunol.0903758

  • CrossRef
  • Google Scholar

• 34

  Luu K Shao Z Schwarz H . The relevance of soluble CD137 in the regulation of immune responses and for immunotherapeutic intervention. J Leukoc Biol. (2020) 107:731–8. doi: 10.1002/JLB.2MR1119-224R

  • CrossRef
  • Google Scholar

• 35

  Thum E Shao Z Schwarz H . CD137, implications in immunity and potential for therapy. Front Biosci (Landmark Ed). (2009) 14:4173–88. doi: 10.2741/3521

  • CrossRef
  • Google Scholar

• 36

  Nielsen C Ohm-Laursen L Barington T Husby S Lillevang ST . Alternative splice variants of the human PD-1 gene. Cell Immunol. (2005) 235:109–16. doi: 10.1016/j.cellimm.2005.07.007

  • CrossRef
  • Google Scholar

• 37

  Blake D Lynch KW . The three as: Alternative splicing, alternative polyadenylation and their impact on apoptosis in immune function. Immunol Rev. (2021) 304:30–50. doi: 10.1111/imr.v304.1

  • CrossRef
  • Google Scholar

• 38

  Jensen LE Whitehead AS . Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis. Cell Signal. (2003) 15:793–802. doi: 10.1016/S0898-6568(03)00039-1

  • CrossRef
  • Google Scholar

• 39

  Oaks MK Hallett KM . Cutting edge: a soluble form of CTLA-4 in patients with autoimmune thyroid disease. J Immunol. (2000) 164:5015–8. doi: 10.4049/jimmunol.164.10.5015

  • CrossRef
  • Google Scholar

• 40

  Grosche L Knippertz I Konig C Royzman D Wild AB Zinser E et al . The CD83 molecule - an important immune checkpoint. Front Immunol. (2020) 11:721. doi: 10.3389/fimmu.2020.00721

  • CrossRef
  • Google Scholar

• 41

  Kuipers H Muskens F Willart M Hijdra D van Assema FB Coyle AJ et al . Contribution of the PD-1 ligands/PD-1 signaling pathway to dendritic cell-mediated CD4+ T cell activation. Eur J Immunol. (2006) 36:2472–82. doi: 10.1002/eji.200635978

  • CrossRef
  • Google Scholar

• 42

  Horvatinovich JM Grogan EW Norris M Steinkasserer A Lemos H Mellor AL et al . Soluble CD83 inhibits T cell activation by binding to the TLR4/MD-2 complex on CD14(+) monocytes. J Immunol. (2017) 198:2286–301. doi: 10.4049/jimmunol.1600802

  • CrossRef
  • Google Scholar

• 43

  Grohmann U Volpi C Fallarino F Bozza S Bianchi R Vacca C et al . Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Nat Med. (2007) 13:579–86. doi: 10.1038/nm1563

  • CrossRef
  • Google Scholar

• 44

  Vardam TD Zhou L Appenheimer MM Chen Q Wang WC Baumann H et al . Regulation of a lymphocyte-endothelial-IL-6 trans-signaling axis by fever-range thermal stress: hot spot of immune surveillance. Cytokine. (2007) 39:84–96. doi: 10.1016/j.cyto.2007.07.184

  • CrossRef
  • Google Scholar

• 45

  Lundstrom W Highfill S Walsh ST Beq S Morse E Kockum I et al . Soluble IL7Ralpha potentiates IL-7 bioactivity and promotes autoimmunity. Proc Natl Acad Sci United States America. (2013) 110:E1761–70. doi: 10.1073/pnas.1222303110

  • CrossRef
  • Google Scholar

• 46

  Aderka D Engelmann H Maor Y Brakebusch C Wallach D . Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors. J Exp Med. (1992) 175:323–9. doi: 10.1084/jem.175.2.323

  • CrossRef
  • Google Scholar

• 47

  Peters M Jacobs S Ehlers M Vollmer P Mullberg J Wolf E et al . The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6. J Exp Med. (1996) 183:1399–406. doi: 10.1084/jem.183.4.1399

  • CrossRef
  • Google Scholar

• 48

  Murphy KM Weaver C . Janeway’s Immunobiology. New York and London: Garland Science/Taylor & Francis Group L (2017) p. 196–7.

  • Google Scholar

• 49

  Kefaloyianni E . Soluble forms of cytokine and growth factor receptors: mechanisms of generation and modes of action in the regulation of local and systemic inflammation. FEBS letters. (2022) 596:589–606. doi: 10.1002/1873-3468.14305

  • CrossRef
  • Google Scholar

• 50

  Dudziak D Nimmerjahn F Bornkamm GW Laux G . Alternative splicing generates putative soluble CD83 proteins that inhibit T cell proliferation. J Immunol. (2005) 174:6672–6. doi: 10.4049/jimmunol.174.11.6672

  • CrossRef
  • Google Scholar

• 51

  Goodwin RG Friend D Ziegler SF Jerzy R Falk BA Gimpel S et al . Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily. Cell. (1990) 60:941–51. doi: 10.1016/0092-8674(90)90342-C

  • CrossRef
  • Google Scholar

• 52

  Cheng J Zhou T Liu C Shapiro JP Brauer MJ Kiefer MC et al . Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science. (1994) 263:1759–62. doi: 10.1126/science.7510905

  • CrossRef
  • Google Scholar

• 53

  Cascino I Fiucci G Papoff G Ruberti G . Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol. (1995) 154:2706–13. doi: 10.4049/jimmunol.154.6.2706

  • CrossRef
  • Google Scholar

• 54

  Horiuchi S Koyanagi Y Zhou Y Miyamoto H Tanaka Y Waki M et al . Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism. Eur J Immunol. (1994) 24:1945–8. doi: 10.1002/eji.1830240837

  • CrossRef
  • Google Scholar

• 55

  Monaghan SF Banerjee D Chung CS Lomas-Neira J Cygan KJ Rhine CL et al . Changes in the process of alternative RNA splicing results in soluble B and T lymphocyte attenuator with biological and clinical implications in critical illness. Mol Med. (2018) 24:32. doi: 10.1186/s10020-018-0036-3

  • CrossRef
  • Google Scholar

• 56

  Nocentini G Ronchetti S Bartoli A Spinicelli S Delfino D Brunetti L et al . Identification of three novel mRNA splice variants of GITR. Cell Death Differ. (2000) 7:408–10. doi: 10.1038/sj.cdd.4400670

  • CrossRef
  • Google Scholar

• 57

  Kruse S Forster J Kuehr J Deichmann KA . Characterization of the membrane-bound and a soluble form of human IL-4 receptor alpha produced by alternative splicing. Int Immunol. (1999) 11:1965–70. doi: 10.1093/intimm/11.12.1965

  • CrossRef
  • Google Scholar

• 58

  Magistrelli G Jeannin P Herbault N Benoit De Coignac A Gauchat JF Bonnefoy JY et al . A soluble form of CTLA-4 generated by alternative splicing is expressed by nonstimulated human T cells. Eur J Immunol. (1999) 29:3596–602. doi: 10.1002/(SICI)1521-4141(199911)29:11<3596::AID-IMMU3596>3.0.CO;2-Y

  • CrossRef
  • Google Scholar

• 59

  Lainez B Fernandez-Real JM Romero X Esplugues E Canete JD Ricart W et al . Identification and characterization of a novel spliced variant that encodes human soluble tumor necrosis factor receptor 2. Int Immunol. (2004) 16:169–77. doi: 10.1093/intimm/dxh014

  • CrossRef
  • Google Scholar

• 60

  Philippe C Roux-Lombard P Fouqueray B Perez J Dayer JM Baud L . Membrane expression and shedding of tumour necrosis factor receptors during activation of human blood monocytes: regulation by desferrioxamine. Immunology. (1993) 80:300–5.

  • Google Scholar

• 61

  Levine SJ . Molecular mechanisms of soluble cytokine receptor generation. J Biol Chem. (2008) 283:14177–81. doi: 10.1074/jbc.R700052200

  • CrossRef
  • Google Scholar

• 62

  Sung HH Juang JH Lin YC Kuo CH Hung JT Chen A et al . Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice. J Exp Med. (2004) 199:1143–51. doi: 10.1084/jem.20031939

  • CrossRef
  • Google Scholar

• 63

  Odales J Guzman Valle J Martinez-Cortes F Manoutcharian K . Immunogenic properties of immunoglobulin superfamily members within complex biological networks. Cell Immunol. (2020) 358:104235. doi: 10.1016/j.cellimm.2020.104235

  • CrossRef
  • Google Scholar

• 64

  Gu D Ao X Yang Y Chen Z Xu X . Soluble immune checkpoints in cancer: production, function and biological significance. J Immunother Cancer. (2018) 6:132. doi: 10.1186/s40425-018-0449-0

  • CrossRef
  • Google Scholar

• 65

  Chakrabarti R Kapse B Mukherjee G . Soluble immune checkpoint molecules: Serum markers for cancer diagnosis and prognosis. Cancer Rep (Hoboken). (2019) 2:e1160. doi: 10.1002/cnr2.1160

  • CrossRef
  • Google Scholar

• 66

  Triebel F . LAG-3: a regulator of T-cell and DC responses and its use in therapeutic vaccination. Trends Immunol. (2003) 24:619–22. doi: 10.1016/j.it.2003.10.001

  • CrossRef
  • Google Scholar

• 67

  Sharpe AH Pauken KE . The diverse functions of the PD1 inhibitory pathway. Nat Rev. (2018) 18:153–67. doi: 10.1038/nri.2017.108

  • CrossRef
  • Google Scholar

• 68

  Keir ME Butte MJ Freeman GJ Sharpe AH . PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. (2008) 26:677–704. doi: 10.1146/annurev.immunol.26.021607.090331

  • CrossRef
  • Google Scholar

• 69

  Song MY Park SH Nam HJ Choi DH Sung YC . Enhancement of vaccine-induced primary and memory CD8(+) T-cell responses by soluble PD-1. J Immunother. (2011) 34:297–306. doi: 10.1097/CJI.0b013e318210ed0e

  • CrossRef
  • Google Scholar

• 70

  He L Zhang G He Y Zhu H Zhang H Feng Z . Blockade of B7-H1 with sPD-1 improves immunity against murine hepatocarcinoma. Anticancer Res. (2005) 25:3309–13.

  • Google Scholar

• 71

  He YF Zhang GM Wang XH Zhang H Yuan Y Li D et al . Blocking programmed death-1 ligand-PD-1 interactions by local gene therapy results in enhancement of antitumor effect of secondary lymphoid tissue chemokine. J Immunol. (2004) 173:4919–28. doi: 10.4049/jimmunol.173.8.4919

  • CrossRef
  • Google Scholar

• 72

  Dostert C Grusdat M Letellier E Brenner D . The TNF family of ligands and receptors: communication modules in the immune system and beyond. Physiol Rev. (2019) 99:115–60. doi: 10.1152/physrev.00045.2017

  • CrossRef
  • Google Scholar

• 73

  Fine A Anderson NL Rothstein TL Williams MC Gochuico BR . Fas expression in pulmonary alveolar type II cells. Am J Physiol. (1997) 273:L64–71. doi: 10.1152/ajplung.1997.273.1.L64

  • CrossRef
  • Google Scholar

• 74

  Pinkoski MJ Brunner T Green DR Lin T . Fas and Fas ligand in gut and liver. Am J Physiol Gastrointest Liver Physiol. (2000) 278:G354–66. doi: 10.1152/ajpgi.2000.278.3.G354

  • CrossRef
  • Google Scholar

• 75

  Krammer PH . CD95’s deadly mission in the immune system. Nature. (2000) 407:789–95. doi: 10.1038/35037728

  • CrossRef
  • Google Scholar

• 76

  Golstein P Griffiths GM . An early history of T cell-mediated cytotoxicity. Nat Rev. (2018) 18:527–35. doi: 10.1038/s41577-018-0009-3

  • CrossRef
  • Google Scholar

• 77

  Klebanoff CA Scott CD Leonardi AJ Yamamoto TN Cruz AC Ouyang C et al . Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J Clin Invest. (2016) 126:318–34. doi: 10.1172/JCI81217

  • CrossRef
  • Google Scholar

• 78

  Sprent J Kishimoto H . The thymus and negative selection. Immunol Rev. (2002) 185:126–35. doi: 10.1034/j.1600-065X.2002.18512.x

  • CrossRef
  • Google Scholar

• 79

  Alderson MR Tough TW Davis-Smith T Braddy S Falk B Schooley KA et al . Fas ligand mediates activation-induced cell death in human T lymphocytes. J Exp Med. (1995) 181:71–7. doi: 10.1084/jem.181.1.71

  • CrossRef
  • Google Scholar

• 80

  Kunkele A Johnson AJ Rolczynski LS Chang CA Hoglund V Kelly-Spratt KS et al . Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenuated due to Cell Fas-FasL-Dependent AICD. Cancer Immunol Res. (2015) 3:368–79. doi: 10.1158/2326-6066.CIR-14-0200

  • CrossRef
  • Google Scholar

• 81

  Horton BL Williams JB Cabanov A Spranger S Gajewski TF . Intratumoral CD8(+) T-cell apoptosis is a major component of T-cell dysfunction and impedes antitumor immunity. Cancer Immunol Res. (2018) 6:14–24. doi: 10.1158/2326-6066.CIR-17-0249

  • CrossRef
  • Google Scholar

• 82

  Izquierdo JM Majos N Bonnal S Martinez C Castelo R Guigo R et al . Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. Mol Cell. (2005) 19:475–84. doi: 10.1016/j.molcel.2005.06.015

  • CrossRef
  • Google Scholar

• 83

  Izquierdo JM Hu antigen R . (HuR) functions as an alternative pre-mRNA splicing regulator of Fas apoptosis-promoting receptor on exon definition. J Biol Chem. (2008) 283:19077–84. doi: 10.1074/jbc.M800017200

  • CrossRef
  • Google Scholar

• 84

  Oh H Lee E Jang HN Lee J Moon H Sheng Z et al . hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene. Apoptosis. (2013) 18:825–35. doi: 10.1007/s10495-013-0824-8

  • CrossRef
  • Google Scholar

• 85

  Jang HN Liu Y Choi N Oh J Ha J Zheng X et al . Binding of SRSF4 to a novel enhancer modulates splicing of exon 6 of Fas pre-mRNA. Biochem Biophys Res Commun. (2018) 506:703–8. doi: 10.1016/j.bbrc.2018.10.123

  • CrossRef
  • Google Scholar

• 86

  Tejedor JR Papasaikas P Valcarcel J . Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis. Mol Cell. (2015) 57:23–38. doi: 10.1016/j.molcel.2014.10.029

  • CrossRef
  • Google Scholar

• 87

  Choi N Jang HN Oh J Ha J Park H Zheng X et al . SRSF6 regulates the alternative splicing of the apoptotic fas gene by targeting a novel RNA sequence. Cancers (Basel). (2022) 14(8):1990. doi: 10.3390/cancers14081990

  • CrossRef
  • Google Scholar

• 88

  Bajgain P Torres Chavez AG Balasubramanian K Fleckenstein L Lulla P Heslop HE et al . Secreted fas decoys enhance the antitumor activity of engineered and bystander T cells in fas ligand-expressing solid tumors. Cancer Immunol Res. (2022) 10:1370–85. doi: 10.1158/2326-6066.CIR-22-0115

  • CrossRef
  • Google Scholar

• 89

  Batlle E Massague J . Transforming growth factor-beta signaling in immunity and cancer. Immunity. (2019) 50:924–40. doi: 10.1016/j.immuni.2019.03.024

  • CrossRef
  • Google Scholar

• 90

  Chakravarthy A Khan L Bensler NP Bose P De Carvalho DD . TGF-beta-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nat Commun. (2018) 9:4692. doi: 10.1038/s41467-018-06654-8

  • CrossRef
  • Google Scholar

• 91

  Tauriello DVF Palomo-Ponce S Stork D Berenguer-Llergo A Badia-Ramentol J Iglesias M et al . TGFbeta drives immune evasion in genetically reconstituted colon cancer metastasis. Nature. (2018) 554:538–43. doi: 10.1038/nature25492

  • CrossRef
  • Google Scholar

• 92

  Ni Y Soliman A Joehlin-Price A Rose PG Vlad A Edwards RP et al . High TGF-beta signature predicts immunotherapy resistance in gynecologic cancer patients treated with immune checkpoint inhibition. NPJ Precis Oncol. (2021) 5:101. doi: 10.1038/s41698-021-00242-8

  • CrossRef
  • Google Scholar

• 93

  Bertolio MS La Colla A Carrea A Romo A Canziani G Echarte SM et al . A novel splice variant of human TGF-beta type II receptor encodes a soluble protein and its fc-tagged version prevents liver fibrosis in vivo. Front Cell Dev Biol. (2021) 9:690397. doi: 10.3389/fcell.2021.690397

  • CrossRef
  • Google Scholar

• 94

  Lust JA Donovan KA Kline MP Greipp PR Kyle RA Maihle NJ . Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor. Cytokine. (1992) 4:96–100. doi: 10.1016/1043-4666(92)90043-Q

  • CrossRef
  • Google Scholar

• 95

  Garapati VP Lefranc MP . IMGT Colliers de Perles and IgSF domain standardization for T cell costimulatory activatory (CD28, ICOS) and inhibitory (CTLA4, PDCD1 and BTLA) receptors. Dev Comp Immunol. (2007) 31:1050–72. doi: 10.1016/j.dci.2007.01.008

  • CrossRef
  • Google Scholar

• 96

  Andrzejczak A Karabon L . BTLA biology in cancer: from bench discoveries to clinical potentials. biomark Res. (2024) 12:8. doi: 10.1186/s40364-024-00556-2

  • CrossRef
  • Google Scholar

• 97

  Magistrelli G Jeannin P Elson G Gauchat JF Nguyen TN Bonnefoy JY et al . Identification of three alternatively spliced variants of human CD28 mRNA. Biochem Biophys Res Commun. (1999) 259:34–7. doi: 10.1006/bbrc.1999.0725

  • CrossRef
  • Google Scholar

• 98

  Hebbar M Jeannin P Magistrelli G Hatron PY Hachulla E Devulder B et al . Detection of circulating soluble CD28 in patients with systemic lupus erythematosus, primary Sjogren’s syndrome and systemic sclerosis. Clin Exp Immunol. (2004) 136:388–92. doi: 10.1111/j.1365-2249.2004.02427.x

  • CrossRef
  • Google Scholar

• 99

  Lechmann M Krooshoop DJ Dudziak D Kremmer E Kuhnt C Figdor CG et al . The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J Exp Med. (2001) 194:1813–21. doi: 10.1084/jem.194.12.1813

  • CrossRef
  • Google Scholar

• 100

  Burnell SEA Capitani L MacLachlan BJ Mason GH Gallimore AM Godkin A . Seven mysteries of LAG-3: a multi-faceted immune receptor of increasing complexity. Immunother Adv. (2022) 2:ltab025. doi: 10.1093/immadv/ltab025

  • CrossRef
  • Google Scholar

• 101

  Gorgulho J Roderburg C Beier F Bokemeyer C Brummendorf TH Loosen SH et al . Soluble lymphocyte activation gene-3 (sLAG3) and CD4/CD8 ratio dynamics as predictive biomarkers in patients undergoing immune checkpoint blockade for solid Malignancies. Br J cancer. (2024) 130:1013–22. doi: 10.1038/s41416-023-02558-7

  • CrossRef
  • Google Scholar

• 102

  Frenay J Bellaye PS Oudot A Helbling A Petitot C Ferrand C et al . IL-1RAP, a key therapeutic target in cancer. Int J Mol Sci. (2022) 23(23):14918. doi: 10.3390/ijms232314918

  • CrossRef
  • Google Scholar

• 103

  Blum H Wolf M Enssle K Rollinghoff M Gessner A . Two distinct stimulus-dependent pathways lead to production of soluble murine interleukin-4 receptor. J Immunol. (1996) 157:1846–53. doi: 10.4049/jimmunol.157.5.1846

  • CrossRef
  • Google Scholar

• 104

  Schumertl T Lokau J Rose-John S Garbers C . Function and proteolytic generation of the soluble interleukin-6 receptor in health and disease. Biochim Biophys Acta Mol Cell Res. (2022) 1869:119143. doi: 10.1016/j.bbamcr.2021.119143

  • CrossRef
  • Google Scholar

• 105

  Hurst SM Wilkinson TS McLoughlin RM Jones S Horiuchi S Yamamoto N et al . Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity. (2001) 14:705–14. doi: 10.1016/S1074-7613(01)00151-0

  • CrossRef
  • Google Scholar

• 106

  Wolf J Waetzig GH Chalaris A Reinheimer TM Wege H Rose-John S et al . Different soluble forms of the interleukin-6 family signal transducer gp130 fine-tune the blockade of interleukin-6 trans-signaling. J Biol Chem. (2016) 291:16186–96. doi: 10.1074/jbc.M116.718551

  • CrossRef
  • Google Scholar

• 107

  Tian J Zhang B Rui K Wang S . The role of GITR/GITRL interaction in autoimmune diseases. Front Immunol. (2020) 11:588682. doi: 10.3389/fimmu.2020.588682

  • CrossRef
  • Google Scholar

• 108

  Romo A Rodriguez TM Yu G Dewey RA . Chimeric TbetaRII-SE/Fc overexpression by a lentiviral vector exerts strong antitumoral activity on colorectal cancer-derived cell lines in vitro and on xenografts. Cancer Gene Ther. (2024) 31:174–85. doi: 10.1038/s41417-023-00694-z

  • CrossRef
  • Google Scholar

• 109

  Fenix AM Miyaoka Y Bertero A Blue SM Spindler MJ Tan KKB et al . Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies. Nat Commun. (2021) 12:6324. doi: 10.1038/s41467-021-26623-y

  • CrossRef
  • Google Scholar

• 110

  Wells QS Becker JR Su YR Mosley JD Weeke P D’Aoust L et al . Whole exome sequencing identifies a causal RBM20 mutation in a large pedigree with familial dilated cardiomyopathy. Circ Cardiovasc Genet. (2013) 6:317–26. doi: 10.1161/CIRCGENETICS.113.000011

  • CrossRef
  • Google Scholar

• 111

  Aartsma-Rus A Van Deutekom JC Fokkema IF Van Ommen GJ Den Dunnen JT . Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. (2006) 34:135–44. doi: 10.1002/mus.20586

  • CrossRef
  • Google Scholar

• 112

  Pagani F Stuani C Tzetis M Kanavakis E Efthymiadou A Doudounakis S et al . New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12. Hum Mol Genet. (2003) 12:1111–20. doi: 10.1093/hmg/ddg131

  • CrossRef
  • Google Scholar

• 113

  Cartegni L Chew SL Krainer AR . Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. (2002) 3:285–98. doi: 10.1038/nrg775

  • CrossRef
  • Google Scholar

• 114

  Pagani F Baralle FE . Genomic variants in exons and introns: identifying the splicing spoilers. Nat Rev Genet. (2004) 5:389–96. doi: 10.1038/nrg1327

  • CrossRef
  • Google Scholar

• 115

  Bergsma AJ van der Wal E Broeders M van der Ploeg AT Pim Pijnappel WWM . Alternative splicing in genetic diseases: improved diagnosis and novel treatment options. Int Rev Cell Mol Biol. (2018) 335:85–141. doi: 10.1016/bs.ircmb.2017.07.008

  • CrossRef
  • Google Scholar

• 116

  Chou J Ohsumi TK Geha RS . Use of whole exome and genome sequencing in the identification of genetic causes of primary immunodeficiencies. Curr Opin Allergy Clin Immunol. (2012) 12:623–8. doi: 10.1097/ACI.0b013e3283588ca6

  • CrossRef
  • Google Scholar

• 117

  Ars E Kruyer H Gaona A Serra E Lazaro C Estivill X . Prenatal diagnosis of sporadic neurofibromatosis type 1 (NF1) by RNA and DNA analysis of a splicing mutation. Prenat Diagn. (1999) 19:739–42. doi: 10.1002/(SICI)1097-0223(199908)19:8<739::AID-PD626>3.0.CO;2-A

  • CrossRef
  • Google Scholar

• 118

  Baralle D Baralle M . Splicing in action: assessing disease causing sequence changes. J Med Genet. (2005) 42:737–48. doi: 10.1136/jmg.2004.029538

  • CrossRef
  • Google Scholar

• 119

  Nik S Bowman TV . Splicing and neurodegeneration: Insights and mechanisms. Wiley Interdiscip Rev RNA. (2019) 10:e1532. doi: 10.1002/wrna.2019.10.issue-4

  • CrossRef
  • Google Scholar

• 120

  Samaranch L Lorenzo-Betancor O Arbelo JM Ferrer I Lorenzo E Irigoyen J et al . PINK1-linked parkinsonism is associated with Lewy body pathology. Brain. (2010) 133:1128–42. doi: 10.1093/brain/awq051

  • CrossRef
  • Google Scholar

• 121

  Fu RH Liu SP Huang SJ Chen HJ Chen PR Lin YH et al . Aberrant alternative splicing events in Parkinson’s disease. Cell Transplant. (2013) 22:653–61. doi: 10.3727/096368912X655154

  • CrossRef
  • Google Scholar

• 122

  Tollervey JR Wang Z Hortobagyi T Witten JT Zarnack K Kayikci M et al . Analysis of alternative splicing associated with aging and neurodegeneration in the human brain. Genome Res. (2011) 21:1572–82. doi: 10.1101/gr.122226.111

  • CrossRef
  • Google Scholar

• 123

  Anderson SL Coli R Daly IW Kichula EA Rork MJ Volpi SA et al . Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet. (2001) 68:753–8. doi: 10.1086/318808

  • CrossRef
  • Google Scholar

• 124

  Licatalosi DD Darnell RB . Splicing regulation in neurologic disease. Neuron. (2006) 52:93–101. doi: 10.1016/j.neuron.2006.09.017

  • CrossRef
  • Google Scholar

• 125

  Li D McIntosh CS Mastaglia FL Wilton SD Aung-Htut MT . Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies. Transl Neurodegener. (2021) 10:16. doi: 10.1186/s40035-021-00240-7

  • CrossRef
  • Google Scholar

• 126

  Holm A Hansen SN Klitgaard H Kauppinen S . Clinical advances of RNA therapeutics for treatment of neurological and neuromuscular diseases. RNA Biol. (2022) 19:594–608. doi: 10.1080/15476286.2022.2066334

  • CrossRef
  • Google Scholar

• 127

  Amoasii L Long C Li H Mireault AA Shelton JM Sanchez-Ortiz E et al . Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Sci Transl Med. (2017) 9(418):eaan8081. doi: 10.1126/scitranslmed.aan8081

  • CrossRef
  • Google Scholar

• 128

  Puttaraju M Jamison SF Mansfield SG Garcia-Blanco MA Mitchell LG . Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat Biotechnol. (1999) 17:246–52. doi: 10.1038/6986

  • CrossRef
  • Google Scholar

• 129

  Rindt H Yen PF Thebeau CN Peterson TS Weisman GA Lorson CL . Replacement of huntingtin exon 1 by trans-splicing. Cell Mol Life Sci. (2012) 69:4191–204. doi: 10.1007/s00018-012-1083-5

  • CrossRef
  • Google Scholar

• 130

  Lorain S Peccate C Le Hir M Griffith G Philippi S Precigout G et al . Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches. Nucleic Acids Res. (2013) 41:8391–402. doi: 10.1093/nar/gkt621

  • CrossRef
  • Google Scholar

• 131

  Avale ME Rodriguez-Martin T Gallo JM . Trans-splicing correction of tau isoform imbalance in a mouse model of tau mis-splicing. Hum Mol Genet. (2013) 22:2603–11. doi: 10.1093/hmg/ddt108

  • CrossRef
  • Google Scholar

• 132

  Shuai S Suzuki H Diaz-Navarro A Nadeu F Kumar SA Gutierrez-Fernandez A et al . The U1 spliceosomal RNA is recurrently mutated in multiple cancers. Nature. (2019) 574:712–6. doi: 10.1038/s41586-019-1651-z

  • CrossRef
  • Google Scholar

• 133

  Suzuki H Kumar SA Shuai S Diaz-Navarro A Gutierrez-Fernandez A De Antonellis P et al . Recurrent noncoding U1 snRNA mutations drive cryptic splicing in SHH medulloblastoma. Nature. (2019) 574:707–11. doi: 10.1038/s41586-019-1650-0

  • CrossRef
  • Google Scholar

• 134

  Yoshida K Sanada M Shiraishi Y Nowak D Nagata Y Yamamoto R et al . Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. (2011) 478:64–9. doi: 10.1038/nature10496

  • CrossRef
  • Google Scholar

• 135

  Graubert TA Shen D Ding L Okeyo-Owuor T Lunn CL Shao J et al . Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. (2011) 44:53–7. doi: 10.1038/ng.1031

  • CrossRef
  • Google Scholar

• 136

  Corrigendum. Comprehensive molecular profiling of lung carcinoma. Can Genome Atlas Res Netw. Nat. (2014) 511(7511):543–50. doi: 10.1038/nature13385

  • CrossRef
  • Google Scholar

• 137

  Bailey P Chang DK Nones K Johns AL Patch AM Gingras MC et al . Genomic analyses identify molecular subtypes of pancreatic cancer. Nature. (2016) 531:47–52. doi: 10.1038/nature16965

  • CrossRef
  • Google Scholar

• 138

  Brooks AN Choi PS de Waal L Sharifnia T Imielinski M Saksena G et al . A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events. PloS One. (2014) 9:e87361. doi: 10.1371/journal.pone.0087361

  • CrossRef
  • Google Scholar

• 139

  Przychodzen B Jerez A Guinta K Sekeres MA Padgett R Maciejewski JP et al . Patterns of missplicing due to somatic U2AF1 mutations in myeloid neoplasms. Blood. (2013) 122:999–1006. doi: 10.1182/blood-2013-01-480970

  • CrossRef
  • Google Scholar

• 140

  Lopez-Canovas JL Del Rio-Moreno M Garcia-Fernandez H Jimenez-Vacas JM Moreno-Montilla MT Sanchez-Frias ME et al . Splicing factor SF3B1 is overexpressed and implicated in the aggressiveness and survival of hepatocellular carcinoma. Cancer Lett. (2021) 496:72–83. doi: 10.1016/j.canlet.2020.10.010

  • CrossRef
  • Google Scholar

• 141

  Papaemmanuil E Cazzola M Boultwood J Malcovati L Vyas P Bowen D et al . Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. New Engl J Med. (2011) 365:1384–95. doi: 10.1056/NEJMoa1103283

  • CrossRef
  • Google Scholar

• 142

  Furney SJ Pedersen M Gentien D Dumont AG Rapinat A Desjardins L et al . SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discovery. (2013) 3:1122–9. doi: 10.1158/2159-8290.CD-13-0330

  • CrossRef
  • Google Scholar

• 143

  Maguire SL Leonidou A Wai P Marchio C Ng CK Sapino A et al . SF3B1 mutations constitute a novel therapeutic target in breast cancer. J pathol. (2015) 235:571–80. doi: 10.1002/path.2015.235.issue-4

  • CrossRef
  • Google Scholar

• 144

  Gentien D Kosmider O Nguyen-Khac F Albaud B Rapinat A Dumont AG et al . A common alternative splicing signature is associated with SF3B1 mutations in Malignancies from different cell lineages. Leukemia. (2014) 28:1355–7. doi: 10.1038/leu.2014.28

  • CrossRef
  • Google Scholar

• 145

  Obeng EA Chappell RJ Seiler M Chen MC Campagna DR Schmidt PJ et al . Physiologic expression of sf3b1(K700E) causes impaired erythropoiesis, aberrant splicing, and sensitivity to therapeutic spliceosome modulation. Cancer Cell. (2016) 30:404–17. doi: 10.1016/j.ccell.2016.08.006

  • CrossRef
  • Google Scholar

• 146

  Zhou Z Gong Q Wang Y Li M Wang L Ding H et al . The biological function and clinical significance of SF3B1 mutations in cancer. biomark Res. (2020) 8:38. doi: 10.1186/s40364-020-00220-5

  • CrossRef
  • Google Scholar

• 147

  Alsafadi S Houy A Battistella A Popova T Wassef M Henry E et al . Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat Commun. (2016) 7:10615. doi: 10.1038/ncomms10615

  • CrossRef
  • Google Scholar

• 148

  Sun G Zhou H Chen K Zeng J Zhang Y Yan L et al . Retraction Note: HnRNP A1 - mediated alternative splicing of CCDC50 contributes to cancer progression of clear cell renal cell carcinoma via ZNF395. J Exp Clin Cancer Res. (2023) 42:43. doi: 10.1186/s13046-023-02613-4

  • CrossRef
  • Google Scholar

• 149

  Cogoi S Rapozzi V Cauci S Xodo LE . Critical role of hnRNP A1 in activating KRAS transcription in pancreatic cancer cells: A molecular mechanism involving G4 DNA. Biochim Biophys Acta Gen Subj. (2017) 1861:1389–98. doi: 10.1016/j.bbagen.2016.11.031

  • CrossRef
  • Google Scholar

• 150

  Chen Y Liu J Wang W Xiang L Wang J Liu S et al . High expression of hnRNPA1 promotes cell invasion by inducing EMT in gastric cancer. Oncol Rep. (2018) 39:1693–701. doi: 10.3892/or.2018.6273

  • CrossRef
  • Google Scholar

• 151

  Ryu HG Jung Y Lee N Seo JY Kim SW Lee KH et al . HNRNP A1 promotes lung cancer cell proliferation by modulating VRK1 translation. Int J Mol Sci. (2021) 22(11):5506. doi: 10.3390/ijms22115506

  • CrossRef
  • Google Scholar

• 152

  Loh TJ Moon H Cho S Jang H Liu YC Tai H et al . CD44 alternative splicing and hnRNP A1 expression are associated with the metastasis of breast cancer. Oncol Rep. (2015) 34:1231–8. doi: 10.3892/or.2015.4110

  • CrossRef
  • Google Scholar

• 153

  Lv Y Zhang W Zhao J Sun B Qi Y Ji H et al . SRSF1 inhibits autophagy through regulating Bcl-x splicing and interacting with PIK3C3 in lung cancer. Signal Transduct Target Ther. (2021) 6:108. doi: 10.1038/s41392-021-00495-6

  • CrossRef
  • Google Scholar

• 154

  Karni R de StanChina E Lowe SW Sinha R Mu D Krainer AR . The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol. (2007) 14:185–93. doi: 10.1038/nsmb1209

  • CrossRef
  • Google Scholar

• 155

  Comiskey DF Jr. Montes M Khurshid S Singh RK Chandler DS . SRSF2 regulation of MDM2 reveals splicing as a therapeutic vulnerability of the p53 pathway. Mol Cancer Res. (2020) 18:194–203. doi: 10.1158/1541-7786.MCR-19-0541

  • CrossRef
  • Google Scholar

• 156

  Lei S Zhang B Huang L Zheng Z Xie S Shen L et al . SRSF1 promotes the inclusion of exon 3 of SRA1 and the invasion of hepatocellular carcinoma cells by interacting with exon 3 of SRA1pre-mRNA. Cell Death Discovery. (2021) 7:117. doi: 10.1038/s41420-021-00498-w

  • CrossRef
  • Google Scholar

• 157

  Liu H Gong Z Li K Zhang Q Xu Z Xu Y . SRPK1/2 and PP1alpha exert opposite functions by modulating SRSF1-guided MKNK2 alternative splicing in colon adenocarcinoma. J Exp Clin Cancer Res. (2021) 40:75. doi: 10.1186/s13046-021-01877-y

  • CrossRef
  • Google Scholar

• 158

  Jiang L Huang J Higgs BW Hu Z Xiao Z Yao X et al . Genomic landscape survey identifies SRSF1 as a key oncodriver in small cell lung cancer. PloS Genet. (2016) 12:e1005895. doi: 10.1371/journal.pgen.1005895

  • CrossRef
  • Google Scholar

• 159

  Anczukow O Akerman M Clery A Wu J Shen C Shirole NH et al . SRSF1-regulated alternative splicing in breast cancer. Mol Cell. (2015) 60:105–17. doi: 10.1016/j.molcel.2015.09.005

  • CrossRef
  • Google Scholar

• 160

  Jensen MA Wilkinson JE Krainer AR . Splicing factor SRSF6 promotes hyperplasia of sensitized skin. Nat Struct Mol Biol. (2014) 21:189–97. doi: 10.1038/nsmb.2756

  • CrossRef
  • Google Scholar

• 161

  Wan L Yu W Shen E Sun W Liu Y Kong J et al . SRSF6-regulated alternative splicing that promotes tumour progression offers a therapy target for colorectal cancer. Gut. (2019) 68:118–29. doi: 10.1136/gutjnl-2017-314983

  • CrossRef
  • Google Scholar

• 162

  Cohen-Eliav M Golan-Gerstl R Siegfried Z Andersen CL Thorsen K Orntoft TF et al . The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers. J pathol. (2013) 229:630–9. doi: 10.1002/path.4129

  • CrossRef
  • Google Scholar

• 163

  Kong-Beltran M Seshagiri S Zha J Zhu W Bhawe K Mendoza N et al . Somatic mutations lead to an oncogenic deletion of met in lung cancer. Cancer Res. (2006) 66:283–9. doi: 10.1158/0008-5472.CAN-05-2749

  • CrossRef
  • Google Scholar

• 164

  Onozato R Kosaka T Kuwano H Sekido Y Yatabe Y Mitsudomi T . Activation of MET by gene amplification or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers. J Thorac Oncol. (2009) 4:5–11. doi: 10.1097/JTO.0b013e3181913e0e

  • CrossRef
  • Google Scholar

• 165

  Schrock AB Frampton GM Suh J Chalmers ZR Rosenzweig M Erlich RL et al . Characterization of 298 patients with lung cancer harboring MET exon 14 skipping alterations. J Thorac Oncol. (2016) 11:1493–502. doi: 10.1016/j.jtho.2016.06.004

  • CrossRef
  • Google Scholar

• 166

  Peschard P Fournier TM Lamorte L Naujokas MA Band H Langdon WY et al . Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein. Mol Cell. (2001) 8:995–1004. doi: 10.1016/S1097-2765(01)00378-1

  • CrossRef
  • Google Scholar

• 167

  Awad MM Lee JK Madison R Classon A Kmak J Frampton GM et al . Characterization of 1,387 NSCLCs with MET exon 14 (METex14) skipping alterations (SA) and potential acquired resistance (AR) mechanisms. J Clin Oncol. (2020) 38. doi: 10.1200/JCO.2020.38.15_suppl.9511

  • CrossRef
  • Google Scholar

• 168

  Heist RS Shim HS Gingipally S Mino-Kenudson M Le L Gainor JF et al . MET exon 14 skipping in non-small cell lung cancer. Oncologist. (2016) 21. doi: 10.1634/theoncologist.2015-0510

  • CrossRef
  • Google Scholar

• 169

  Gorlov IP Gorlova OY Frazier ML Amos CI . Missense mutations in hMLH1 and hMSH2 are associated with exonic splicing enhancers. Am J Hum Genet. (2003) 73:1157–61. doi: 10.1086/378819

  • CrossRef
  • Google Scholar

• 170

  Liu T Tannergard P Hackman P Rubio C Kressner U Lindmark G et al . Missense mutations in hMLH1 associated with colorectal cancer. Hum Genet. (1999) 105:437–41. doi: 10.1007/s004399900160

  • CrossRef
  • Google Scholar

• 171

  Wijnen J Khan PM Vasen H Menko F van der Klift H van den Broek M et al . Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16. Am J Hum Genet. (1996) 58:300–7.

  • Google Scholar

• 172

  Stella A Wagner A Shito K Lipkin SM Watson P Guanti G et al . A nonsense mutation in MLH1 causes exon skipping in three unrelated HNPCC families. Cancer Res. (2001) 61:7020–4.

  • Google Scholar

• 173

  Clarke LA Veiga I Isidro G Jordan P Ramos JS Castedo S et al . Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation. Genes Chromosomes Cancer. (2000) 29:367–70. doi: 10.1002/1098-2264(2000)9999:9999<::AID-GCC1051>3.0.CO;2-V

  • CrossRef
  • Google Scholar

• 174

  Tanko Q Franklin B Lynch H Knezetic J . A hMLH1 genomic mutation and associated novel mRNA defects in a hereditary non-polyposis colorectal cancer family. Mutat Res. (2002) 503:37–42. doi: 10.1016/S0027-5107(02)00031-3

  • CrossRef
  • Google Scholar

• 175

  Smeby J Sveen A Eilertsen IA Danielsen SA Hoff AM Eide PW et al . Transcriptional and functional consequences of TP53 splice mutations in colorectal cancer. Oncogenesis. (2019) 8:35. doi: 10.1038/s41389-019-0141-3

  • CrossRef
  • Google Scholar

• 176

  Bodner SM Minna JD Jensen SM D’Amico D Carbone D Mitsudomi T et al . Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation. Oncogene. (1992) 7:743–9.

  • Google Scholar

• 177

  Surget S Khoury MP Bourdon JC . Uncovering the role of p53 splice variants in human Malignancy: a clinical perspective. Onco Targets Ther. (2013) 7:57–68. doi: 10.2147/OTT.S53876

  • CrossRef
  • Google Scholar

• 178

  Bourdon JC . p53 and its isoforms in cancer. Br J cancer. (2007) 97:277–82. doi: 10.1038/sj.bjc.6603886

  • CrossRef
  • Google Scholar

• 179

  Leroy B Anderson M Soussi T . TP53 mutations in human cancer: database reassessment and prospects for the next decade. Hum Mutat. (2014) 35:672–88. doi: 10.1002/humu.2014.35.issue-6

  • CrossRef
  • Google Scholar

• 180

  Bamopoulos SA Batcha AMN Jurinovic V Rothenberg-Thurley M Janke H Ksienzyk B et al . Clinical presentation and differential splicing of SRSF2, U2AF1 and SF3B1 mutations in patients with acute myeloid leukemia. Leukemia. (2020) 34:2621–34. doi: 10.1038/s41375-020-0839-4

  • CrossRef
  • Google Scholar

• 181

  Martin M Masshofer L Temming P Rahmann S Metz C Bornfeld N et al . Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet. (2013) 45:933–6. doi: 10.1038/ng.2674

  • CrossRef
  • Google Scholar

• 182

  Group PTC Calabrese C Davidson NR Demircioglu D Fonseca NA He Y et al . Genomic basis for RNA alterations in cancer. Nature. (2020) 578:129–36. doi: 10.1038/s41586-020-1970-0

  • CrossRef
  • Google Scholar

• 183

  Puttick C Jones TP Leung MM Galvez-Cancino F Liu J Varas-Godoy M et al . MHC Hammer reveals genetic and non-genetic HLA disruption in cancer evolution. Nat Genet. (2024) 56:2121–31. doi: 10.1038/s41588-024-01883-8

  • CrossRef
  • Google Scholar

• 184

  Wei L Li Y Chen J Wang Y Wu J Yang H et al . Alternative splicing in ovarian cancer. Cell Commun Signal. (2024) 22:507. doi: 10.1186/s12964-024-01880-8

  • CrossRef
  • Google Scholar

• 185

  Cho N Kim SY Lee SG Park C Choi S Kim EM et al . Alternative splicing of PBRM1 mediates resistance to PD-1 blockade therapy in renal cancer. EMBO J. (2024) 43(22):5421–44. doi: 10.1038/s44318-024-00262-7

  • CrossRef
  • Google Scholar

• 186

  Lu SX De Neef E Thomas JD Sabio E Rousseau B Gigoux M et al . Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Cell. (2021) 184:4032–47 e31. doi: 10.1016/j.cell.2021.05.038

  • CrossRef
  • Google Scholar

• 187

  Carvalho T Martins S Rino J Marinho S Carmo-Fonseca M . Pharmacological inhibition of the spliceosome subunit SF3b triggers exon junction complex-independent nonsense-mediated decay. J Cell sci. (2017) 130:1519–31. doi: 10.1242/jcs.202200

  • CrossRef
  • Google Scholar

• 188

  Mahoney KM Shukla SA Patsoukis N Chaudhri A Browne EP Arazi A et al . A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression. Cancer Immunol Immunother. (2019) 68:421–32. doi: 10.1007/s00262-018-2282-1

  • CrossRef
  • Google Scholar

• 189

  Jain M Abu-Shumays R Olsen HE Akeson M . Advances in nanopore direct RNA sequencing. Nat Methods. (2022) 19:1160–4. doi: 10.1038/s41592-022-01633-w

  • CrossRef
  • Google Scholar

• 190

  Krizanovic K Echchiki A Roux J Sikic M . Evaluation of tools for long read RNA-seq splice-aware alignment. Bioinformatics. (2018) 34:748–54. doi: 10.1093/bioinformatics/btx668

  • CrossRef
  • Google Scholar

• 191

  Mehmood A Laiho A Venalainen MS McGlinchey AJ Wang N Elo LL . Systematic evaluation of differential splicing tools for RNA-seq studies. Brief Bioinform. (2020) 21:2052–65. doi: 10.1093/bib/bbz126

  • CrossRef
  • Google Scholar

• 192

  Chhangawala S Rudy G Mason CE Rosenfeld JA . The impact of read length on quantification of differentially expressed genes and splice junction detection. Genome Biol. (2015) 16:131. doi: 10.1186/s13059-015-0697-y

  • CrossRef
  • Google Scholar

• 193

  Ziegenhain C Vieth B Parekh S Reinius B Guillaumet-Adkins A Smets M et al . Comparative analysis of single-cell RNA sequencing methods. Mol Cell. (2017) 65:631–43 e4. doi: 10.1016/j.molcel.2017.01.023

  • CrossRef
  • Google Scholar

• 194

  Song Y Botvinnik OB Lovci MT Kakaradov B Liu P Xu JL et al . Single-cell alternative splicing analysis with expedition reveals splicing dynamics during neuron differentiation. Mol Cell. (2017) 67:148–61 e5. doi: 10.1016/j.molcel.2017.06.003

  • CrossRef
  • Google Scholar

• 195

  Illumina . Available online at: https://emea.support.illumina.com/bulletins/2020/04/maximum-read-length-for-illumina-sequencing-platforms.html.

• 196

  Available online at: https://nanoporetech.com/resource-centre/application-note/isoform-detection-single-cell-and-spatial-resolution.

• 197

  Heywood SM Kennedy DS . Purification of myosin translational control RNA and its interaction with myosin messenger RNA. Biochemistry. (1976) 15:3314–9. doi: 10.1021/bi00660a023

  • CrossRef
  • Google Scholar

• 198

  Bester AJ Kennedy DS Heywood SM . Two classes of translational control RNA: their role in the regulation of protein synthesis. Proc Natl Acad Sci U S A. (1975) 72:1523–7. doi: 10.1073/pnas.72.4.1523

  • CrossRef
  • Google Scholar

• 199

  Paterson BM Roberts BE Kuff EL . Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. (1977) 74:4370–4. doi: 10.1073/pnas.74.10.4370

  • CrossRef
  • Google Scholar

• 200

  Zamecnik PC Stephenson ML . Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A. (1978) 75:280–4. doi: 10.1073/pnas.75.1.280

  • CrossRef
  • Google Scholar

• 201

  Stephenson ML Zamecnik PC . Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. (1978) 75:285–8. doi: 10.1073/pnas.75.1.285

  • CrossRef
  • Google Scholar

• 202

  Donis-Keller H . Site specific enzymatic cleavage of RNA. Nucleic Acids Res. (1979) 7:179–92. doi: 10.1093/nar/7.1.179

  • CrossRef
  • Google Scholar

• 203

  Sinha ND Biernat J McManus J Koster H . Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. (1984) 12:4539–57. doi: 10.1093/nar/12.11.4539

  • CrossRef
  • Google Scholar

• 204

  Huang Y Knouse KW Qiu S Hao W Padial NM Vantourout JC et al . A P(V) platform for oligonucleotide synthesis. Science. (2021) 373(6560):1265–70. doi: 10.1126/science.abi9727

  • CrossRef
  • Google Scholar

• 205

  Tamm I Dorken B Hartmann G . Antisense therapy in oncology: new hope for an old idea? Lancet. (2001) 358:489–97. doi: 10.1016/S0140-6736(01)05629-X

  • CrossRef
  • Google Scholar

• 206

  Martin Egli MM . Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. (2023) 51:2529–73. doi: 10.1093/nar/gkad067

  • CrossRef
  • Google Scholar

• 207

  Mourich DV Oda SK Schnell FJ Crumley SL Hauck LL Moentenich CA et al . Alternative splice forms of CTLA-4 induced by antisense mediated splice-switching influences autoimmune diabetes susceptibility in NOD mice. Nucleic Acid Ther. (2014) 24:114–26. doi: 10.1089/nat.2013.0449

  • CrossRef
  • Google Scholar

• 208

  Graziewicz MA Tarrant TK Buckley B Roberts J Fulton L Hansen H et al . An endogenous TNF-alpha antagonist induced by splice-switching oligonucleotides reduces inflammation in hepatitis and arthritis mouse models. Mol Ther. (2008) 16:1316–22. doi: 10.1038/mt.2008.85

  • CrossRef
  • Google Scholar

• 209

  Bauman J Jearawiriyapaisarn N Kole R . Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides. (2009) 19:1–13. doi: 10.1089/oli.2008.0161

  • CrossRef
  • Google Scholar

• 210

  Mendell JR Goemans N Lowes LP Alfano LN Berry K Shao J et al . Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy. Ann Neurol. (2016) 79:257–71. doi: 10.1002/ana.24555

  • CrossRef
  • Google Scholar

• 211

  Crooke ST Vickers TA Lima WF Wu H-J . Antisense Drug Technology: Principles, Strategies, and Applications. CrookeST, editor. Boca Raton, Fla., United States: CRC Press (2008) p. 3–46.

  • Google Scholar

• 212

  Swayze EE Bhat B . Antisense Drug Technology: Principles, Strategies, and Applications. CrookeST, editor. Boca Raton, Fla., United States: CRC Press (2008) p. 143–82.

  • Google Scholar

• 213

  Henry S . Antisense Drug Technology-Principles, Strategies, and Applications. CrookeST, editor. Boca Raton, Fla., United States: CRC Press (2008) p. 327–64.

  • Google Scholar

• 214

  Gleave ME Monia BP . Antisense therapy for cancer. Nat Rev Cancer. (2005) 5:468–79. doi: 10.1038/nrc1631

  • CrossRef
  • Google Scholar

• 215

  Eckstein F . Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. (2014) 24:374–87. doi: 10.1089/nat.2014.0506

  • CrossRef
  • Google Scholar

• 216

  Sands H Gorey-Feret LJ Cocuzza AJ Hobbs FW Chidester D Trainor GL . Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol. (1994) 45:932–43.

  • Google Scholar

• 217

  Bennett CF Swayze EE . RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol. (2010) 50:259–93. doi: 10.1146/annurev.pharmtox.010909.105654

  • CrossRef
  • Google Scholar

• 218

  Wang S Allen N Vickers TA Revenko AS Sun H Liang XH et al . Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides. Nucleic Acids Res. (2018) 46:3579–94. doi: 10.1093/nar/gky145

  • CrossRef
  • Google Scholar

• 219

  Brown DA Kang SH Gryaznov SM DeDionisio L Heidenreich O Sullivan S et al . Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem. (1994) 269:26801–5. doi: 10.1016/S0021-9258(18)47090-1

  • CrossRef
  • Google Scholar

• 220

  Iversen PL . Antisense Drug Technology. CrookeST, editor. Boca Raton, FL: CRC Press (2008) p. 565–82.

  • Google Scholar

• 221

  Summerton J . Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta. (1999) 1489:141–58. doi: 10.1016/S0167-4781(99)00150-5

  • CrossRef
  • Google Scholar

• 222

  Geary RS . Antisense oligonucleotide pharmacokinetics and metabolism. Expert Opin Drug Metab Toxicol. (2009) 5:381–91. doi: 10.1517/17425250902877680

  • CrossRef
  • Google Scholar

• 223

  Langner HK Jastrzebska K Caruthers MH . Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras. J Am Chem Soc. (2020) 142:16240–53. doi: 10.1021/jacs.0c04335

  • CrossRef
  • Google Scholar

• 224

  Finkel RS Mercuri E Darras BT Connolly AM Kuntz NL Kirschner J et al . Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N Engl J Med. (2017) 377:1723–32. doi: 10.1056/NEJMoa1702752

  • CrossRef
  • Google Scholar

• 225

  De Vivo DC Hwu W-L Reyna SP Farwell W Gheuens S Sun P et al . Interim efficacy and safety results from the phase 2 NURTURE study evaluating nusinersen in presymptomatic infants with spinal muscular atrophy. Neurology. (2017) 2017:S46.003.

  • Google Scholar

• 226

  Duan D Goemans N Takeda S Mercuri E Aartsma-Rus A . Duchenne muscular dystrophy. Nat Rev Dis Primers. (2021) 7:13. doi: 10.1038/s41572-021-00248-3

  • CrossRef
  • Google Scholar

• 227

  Lim KR Maruyama R Yokota T . Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Des Devel Ther. (2017) 11:533–45. doi: 10.2147/DDDT.S97635

  • CrossRef
  • Google Scholar

• 228

  Heo YA . Golodirsen: first approval. Drugs. (2020) 80:329–33. doi: 10.1007/s40265-020-01267-2

  • CrossRef
  • Google Scholar

• 229

  Dhillon S . Viltolarsen: first approval. Drugs. (2020) 80:1027–31. doi: 10.1007/s40265-020-01339-3

  • CrossRef
  • Google Scholar

• 230

  Shirley M . Casimersen: first approval. Drugs. (2021) 81:875–9. doi: 10.1007/s40265-021-01512-2

  • CrossRef
  • Google Scholar

• 231

  Crooke ST Liang XH Baker BF Crooke RM . Antisense technology: A review. J Biol Chem. (2021) 296:100416. doi: 10.1016/j.jbc.2021.100416

  • CrossRef
  • Google Scholar

• 232

  Yang R Feng X Arias-Cavieres A Mitchell RM Polo A Hu K et al . Upregulation of SYNGAP1 expression in mice and human neurons by redirecting alternative splicing. Neuron. (2023) 111:1637–50 e5. doi: 10.1016/j.neuron.2023.02.021

  • CrossRef
  • Google Scholar

• 233

  Yuan Y Lopez-Santiago L Denomme N Chen C O’Malley HA Hodges SL et al . ASO restores excitability, GABA signalling and sodium current density in a model of Dravet syndrome. Brain. (2024) 147(4):1231–46. doi: 10.1093/brain/awad349

  • CrossRef
  • Google Scholar

• 234

  Kim H Lenoir S Helfricht A Jung T Karneva ZK Lee Y et al . A pathogenic proteolysis-resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function. JCI Insight. (2022) 7(17):e154108. doi: 10.1172/jci.insight.154108

  • CrossRef
  • Google Scholar

• 235

  Shah S Sharp KJ Raju Ponny S Lee J Watts JK Berry-Kravis E et al . Antisense oligonucleotide rescue of CGG expansion-dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. Proc Natl Acad Sci U S A. (2023) 120:e2302534120. doi: 10.1073/pnas.2302534120

  • CrossRef
  • Google Scholar

• 236

  Oren YS Avizur-BarChad O Ozeri-Galai E Elgrabli R Schirelman MR Blinder T et al . Antisense oligonucleotide splicing modulation as a novel Cystic Fibrosis therapeutic approach for the W1282X nonsense mutation. J cystic fibrosis: Off J Eur Cystic Fibrosis Society. (2022) 21:630–6. doi: 10.1016/j.jcf.2021.12.012

  • CrossRef
  • Google Scholar

• 237

  Michaels WE Pena-Rasgado C Kotaria R Bridges RJ Hastings ML . Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis. Proc Natl Acad Sci U S A. (2022) 119(3):e2114886119. doi: 10.1073/pnas.2114886119

  • CrossRef
  • Google Scholar

• 238

  Dewaele M Tabaglio T Willekens K Bezzi M Teo SX Low DH et al . Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth. J Clin Invest. (2016) 126:68–84. doi: 10.1172/JCI82534

  • CrossRef
  • Google Scholar

• 239

  Oh DY Bang YJ . HER2-targeted therapies - a role beyond breast cancer. Nat Rev Clin Oncol. (2020) 17:33–48. doi: 10.1038/s41571-019-0268-3

  • CrossRef
  • Google Scholar

• 240

  Wan J Sazani P Kole R . Modification of HER2 pre-mRNA alternative splicing and its effects on breast cancer cells. Int J Cancer. (2009) 124:772–7. doi: 10.1002/ijc.v124:4

  • CrossRef
  • Google Scholar

• 241

  Pankratova S Nielsen BN Shiraishi T Nielsen PE . PNA-mediated modulation and redirection of Her-2 pre-mRNA splicing: specific skipping of erbB-2 exon 19 coding for the ATP catalytic domain. Int J Oncol. (2010) 36:29–38.

  • Google Scholar

• 242

  Khurshid S Montes M Comiskey DF Jr. Shane B Matsa E Jung F et al . Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma. NPJ Precis Oncol. (2022) 6:1. doi: 10.1038/s41698-021-00245-5

  • CrossRef
  • Google Scholar

• 243

  Wang Z Jeon HY Rigo F Bennett CF Krainer AR . Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides. Open Biol. (2012) 2:120133. doi: 10.1098/rsob.120133

  • CrossRef
  • Google Scholar

• 244

  (2023). Available online at: https://nanoporetech.com/about-us/news/oxford-nanopore-announces-breakthrough-performance-simplex-single-molecule-accuracy:~:text=They%20detailed%20breakthrough%20performance%20in,enzyme%20engineering%20and%20improved%20models.

  • Google Scholar