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Schematic representation of different splicing-targeting strategies for gene modification. (A) siRNA-, ASO-, and SSO-based strategies (a, b, and c in the figure, respectively) for pre-mRNA editing. (B) Bifunctional oligonucleotides contain one small sequence complementary to the pre-mRNA (targeting domain) and another region (effector domain) that recruits specific regulatory factors to modulate the splicing outcome (TOSS and TOES). (C) Three types of SMaRT approaches are depicted: 5′ trans-splicing, 3′ trans-splicing, and IER, which target the 5′ end, 3′ end, or internal portion of a mutated target pre-mRNA, respectively. See the text for further details on these RNA splicing-editing mechanisms. (D) Schematic representation of the general strategy for correction of splicing defects using modified snRNAs. The cartoon shows the restoration of a mutated 5′ SS of an intron using a modified U1 snRNA.
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Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, includ...
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... Cancer is a deadly disease, and it is anticipated that by 2030, there will be 21 million new cases of the disease 62 . By synthesizing innovative medications with high therapeutic potential, scientists are putting great effort into overcoming this huge menace 63,64 ...
The current research aimed to study the green synthesis of silver oxide nanoparticles (AgONPs) using Rhynchosia capitata (RC) aqueous extract as a potent reducing and stabilizing agent. The obtained RC-AgONPs were characterized using UV, FT-IR, XRD, DLS, SEM, and EDX to investigate the morphology, size, and elemental composition. The size of the RC-AgONPs was found to be ~21.66 nm and an almost uniform distribution was executed by XRD analysis. In vitro studies were performed to reveal biological potential. The AgONPs exhibited efficient DPPH free radical scavenging potential (71.3%), reducing power (63.8 ± 1.77%), and total antioxidant capacity (88.5 ± 4.8%) to estimate their antioxidative power. Antibacterial and antifungal potentials were evaluated using the disc diffusion method against various bacterial and fungal strains, and the zones of inhibition (ZOI) were determined. A brine shrimp cytotoxicity assay was conducted to measure the cytotoxicity potential (LC50: 2.26 μg/mL). In addition, biocompatibility tests were performed to evaluate the biocompatible nature of RC-AgONPs using red blood cells, HEK, and VERO cell lines (< 200 μg/mL). An alpha-amylase inhibition assay was carried out with 67.6 % inhibition. Moreover, In vitro, anticancer activity was performed against Hep-2 liver cancer cell lines, and an LC50 value of 45.94 μg/mL was achieved. Overall, the present study has demonstrated that the utilization of Rhynchosia capitata extract for the biosynthesis of AgONPs offers a cost-effective, eco-friendly, and forthright alternative to traditional approaches for silver nanoparticle synthesis. The RC-AgONPs obtained exhibited significant bioactive properties, positioning them as promising candidates for diverse applications in the spheres of medicine and beyond.
... The SMaRT correction system requires the target mRNA, the spliceosome machinery, and the pre-transsplicing molecule (PTM). 87 Splicing occurs in a series of reactions catalysed by the spliceosome. The major spliceosome comprises U1, U2, U4, U5 and U6 snRNPs (U2-dependent) and target U2-type introns in pre-mRNAs. ...
RNA splicing is an important RNA processing step required by multiexon protein-coding mRNAs and some noncoding RNAs. Precise RNA splicing is required for maintaining gene and cell function; however, mis-spliced RNA transcripts can lead to loss- or gain-of-function effects in human diseases. Mis-spliced RNAs induced by gene mutations or the dysregulation of splicing regulators may result in frameshifts, nonsense-mediated decay (NMD), or inclusion/exclusion of exons. Genetic animal models have characterised multiple splicing factors required for cardiac development or function. Moreover, sarcomeric and ion channel genes, which are closely associated with cardiovascular function and disease, are hotspots for AS. Here, we summarise splicing factors and their targets that are associated with cardiovascular diseases, introduce some therapies potentially related to pathological AS targets, and raise outstanding questions and future directions in this field.
... Along with the two-post transcriptional modification; 5′ capping and 3′ poly A tailing, splicing occurs in a highly coordinated manner to produce a fully functional messenger RNA (mRNA) ready to be translated into the protein of interest via the cytoplasmic ribosomes. Mammalian spliceosomes consist of five essential uridine-rich small nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs), which recruit a massive number of auxiliary splicing factors (~200 proteins) needed for initiating the splicing reaction (Rappsilber et al., 2002;Valadkhan et al., 2009;Suñé-Pou et al., 2020). Spliceosomes drive the alternative splicing (AS), which is a critical regulatory process contributing to the vast diversity of translated proteins in mammalian cells. ...
... The increased demands to treat spliceopathies have ignited the development of innovative therapeutic approaches such as spliceosome-mediated RNA trans-splicing (SMaRT) (Wally et al., 2012), splice switching oligonucleotides (SSO) (Havens and Hastings, 2016), CRISPR/Cas9 (Yuan et al., 2018) and nanomedicine (Garcia-Blanco, 2003;Havens et al., 2013;Suñé-Pou et al., 2017;Suñé-Pou et al., 2020). For decades, scientists have been in route to develop treatments for mis-spliced transcripts using different re-engineered genetic tools such as group I introns ribozymes. ...
Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.
... polypyrimidine (Py) tract. In addition to these core splice-site motifs, other cis-regulatory elements that recruit specific RNA-binding proteins that either activate or repress the use of adjacent splice-sites contribute to the fine-tuning and specificity of this pre-mRNA processing event [5,6]. ...
Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s—ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5′ splice-site (5′ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.
... Additionally, cutting-edge delivery technologies that precisely target cancer cells while preventing an immune system reaction are also in development. Liposomes, polymeric nanoparticles, and exosomes are only a few examples of nanoparticle-based drug delivery techniques already in use because they efficiently protect siRNA molecules by encapsulating them from deterioration [95]. These methods can be more accurate while minimizing negative effects on healthy tissues by adding targeted ligands like peptides or antibodies. ...
Advancements in the clinical applications of small interfering RNA (siRNA) in cancer therapy have opened up new possibilities for precision medicine. siRNAs, as powerful genetic tools, have shown potential in targeting and suppressing the expression of specific genes associated with cancer progression. Their effectiveness has been further enhanced by incorporating them into nanoparticles, which protect siRNAs from degradation and enable targeted delivery. However, despite these promising developments, several challenges persist in the clinical translation of siRNA-based cancer therapy. This comprehensive review explores the progress and challenges associated with the clinical applications of siRNA in cancer therapy. This review highlights the use of siRNA-loaded nanoparticles as an effective delivery system for optimizing siRNA efficacy in various types of carcinomas and the potential of siRNA-based therapy as a genetic approach to overcome limitations associated with conventional chemotherapeutic agents, including severe drug toxicities and organ damage. Moreover, it emphasizes on the key challenges, including off-target effects, enzymatic degradation of siRNAs in serum, low tumor localization, stability issues, and rapid clearance from circulation that need to be addressed for successful clinical development of siRNA-based cancer therapy. Despite these challenges, the review identifies significant avenues for advancing siRNA technology from the laboratory to clinical settings. The ongoing progress in siRNA-loaded nanoparticles for cancer treatment demonstrates potential antitumor activities and safety profiles. By understanding the current state of siRNA-based therapy and addressing the existing challenges, we aim to pave the way for translating siRNA technology into effective oncologic clinics as an improved treatment options for cancer patients.
... In addition to AON, various technologies are in development to harness the approaches. Bifunctional oligonucleotides are made of an antisense portion targeting a specific sequence and a nonhybridizing tail that recruits acting factors to modulate splicing outcome, silencing (TOSS, targeted oligonucleotide silencer of splicing) or enhancing (TOES, targeted oligonucleotide enhancer of splicing) [84,85]. ...
The marketing approval, about ten years ago, of the first disease modulator for patients with cystic fibrosis harboring specific CFTR genotypes (~5% of all patients) brought new hope for their treatment. To date, several therapeutic strategies have been approved and the number of CFTR mutations targeted by therapeutic agents is increasing. Although these drugs do not reverse the existing disease, they help to increase the median life expectancy. However, on the basis of their CFTR genotype, ~10% of patients presently do not qualify for any of the currently available CFTR modulator therapies, particularly patients with splicing mutations (~12% of the reported CFTR mutations). Efforts are currently made to develop therapeutic agents that target disease-causing CFTR variants that affect splicing. This highlights the need to fully identify them by scanning non-coding regions and systematically determine their functional consequences. In this review, we present some examples of CFTR alterations that affect splicing events and the different therapeutic options that are currently developed and tested for splice switching.
... Dystrophin exon 51 is the target of eteplirsen, the only FDA-approved exon-skipping drug; however, there are other drugs designed to target different dystrophin exons in development currently. These drugs are expensive, require repeated administration, and, while they seem well tolerated, their long-term effects are not known [56]. Therefore, new strategies for immunosuppression with anti-fibrosis functions are essential. ...
Duchenne muscular dystrophy is an X-linked disease afflicting 1 in 3500 males that is characterized by muscle weakness and wasting during early childhood, and loss of ambulation and death by early adulthood. Chronic inflammation due to myofiber instability leads to fibrosis, which is a primary cause of loss of ambulation and cardiorespiratory insufficiency. Current standard of care focuses on reducing inflammation with corticosteroids, which have serious adverse effects. It is imperative to identify alternate immunosuppressants as treatments to reduce fibrosis and mortality. Serp-1, a Myxoma virus-derived 55 kDa secreted glycoprotein, has proven efficacy in a range of animal models of acute inflammation, and its safety and efficacy has been shown in a clinical trial. In this initial study, we examined whether pegylated Serp-1 (PEGSerp-1) treatment would ameliorate chronic inflammation in a mouse model for Duchenne muscular dystrophy. Our data revealed a significant reduction in diaphragm fibrosis and increased myofiber diameter, and significantly decreased pro-inflammatory M1 macrophage infiltration. The M2a macrophage and overall T cell populations showed no change. These data demonstrate that treatment with this new class of poxvirus-derived immune-modulating serpin has potential as a therapeutic approach designed to ameliorate DMD pathology and facilitate muscle regeneration.
... AS is a process through which a single precursor mRNA can generate a number of alternative mRNAs, thereby allowing for considerable proteomic diversity and complexity [20,21]. It is currently estimated that nearly 95% of human multi-exonic genes are alternatively spliced, thus giving rise to different protein isoforms. ...
... The second major approach for the modulation of the splicing process, both in vitro and in vivo, is the use of synthetic U1 snRNAs designed to recognize mutant 5′ss, thus restoring complementarity. The first step of the splicing process requires the 5′ end of the U1 snRNA to interact by complementarity with the moderately conserved sequence of the 5′ss [20]. This implies that any mutation in this site may compromise the binding of the U1 snRNA and prevent spliceosome assembly, thus inhibiting the subsequent splicing process. ...
... The second major approach for the modulation of the splicing process, both in vitro and in vivo, is the use of synthetic U1 snRNAs designed to recognize mutant 5 ss, thus restoring complementarity. The first step of the splicing process requires the 5 end of the U1 snRNA to interact by complementarity with the moderately conserved sequence of the 5 ss [20]. This implies that any mutation in this site may compromise the binding of the U1 snRNA and prevent spliceosome assembly, thus inhibiting the subsequent splicing process. ...
Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.
... Targeting splicing mechanisms represents an emerging therapeutic possibility for several human diseases, including cancer [157,197,198]. Common approaches include RNA editing through single-stranded DNA molecules and the use of small-molecule compounds acting on splicing factors' functions [198]. ...
... Targeting splicing mechanisms represents an emerging therapeutic possibility for several human diseases, including cancer [157,197,198]. Common approaches include RNA editing through single-stranded DNA molecules and the use of small-molecule compounds acting on splicing factors' functions [198]. Antisense oligonucleotides (ASOs), called spliceswitching oligonucleotides (SSOs), promote exon skipping or inclusion by binding to the complementary target pre-mRNAs [198]. ...
... Common approaches include RNA editing through single-stranded DNA molecules and the use of small-molecule compounds acting on splicing factors' functions [198]. Antisense oligonucleotides (ASOs), called spliceswitching oligonucleotides (SSOs), promote exon skipping or inclusion by binding to the complementary target pre-mRNAs [198]. In GBM, these strategies have been used to impair the splicing outcomes of the two oncogenic proteins MAPK interacting serine/threonine kinase 2 (MNK2) and human telomerase reverse transcriptase (hTERT), inhibiting the junctions and thus hindering tumour growth [199,200]. ...
The advance of experimental and computational techniques has allowed us to highlight the existence of numerous different mechanisms of RNA maturation, which have been so far unknown. Besides canonical splicing, consisting of the removal of introns from pre-mRNA molecules, non-canonical splicing events may occur to further increase the regulatory and coding potential of the human genome. Among these, splicing of microexons, recursive splicing and biogenesis of circular and chimeric RNAs through back-splicing and trans-splicing processes, respectively, all contribute to expanding the repertoire of RNA transcripts with newly acquired regulatory functions. Interestingly, these non-canonical splicing events seem to occur more frequently in the central nervous system, affecting neuronal development and differentiation programs with important implications on brain physiology. Coherently, dysregulation of non-canonical RNA processing events is associated with brain disorders, including brain tumours. Herein, we summarize the current knowledge on molecular and regulatory mechanisms underlying canonical and non-canonical splicing events with particular emphasis on cis-acting elements and trans-acting factors that all together orchestrate splicing catalysis reactions and decisions. Lastly, we review the impact of non-canonical splicing on brain physiology and pathology and how unconventional splicing mechanisms may be targeted or exploited for novel therapeutic strategies in cancer.
... Obviously, in all the above cases, finding an efficient method to deliver miRNAs or their related molecules is an appealing challenge. Nanotechnology-based gene delivery is a promising method to reach that goal [153]. ...
Simple Summary
Epigenetics studies the alteration of gene expression without changing DNA sequence and very often, epigenetic dysregulation causes cancer. Alternative splicing is a mechanism that results in the production of several mRNA isoforms from a single gene and aberrant splicing is also a frequent cause of cancer. The present review is built on the interrelations of epigenetics and alternative splicing. In an intuitive way, we say that epigenetic modifications and alternative splicing are at two vertices of a triangle, the third vertex being occupied by cancer. Interconnection between alternative splicing and epigenetic modifications occurs backward and forward and the mechanisms involved are widely reviewed. These connections also provide novel diagnostic or prognostic tools, which are listed. Finally, as epigenetic alterations are reversible and aberrant alternative splicing may be corrected, the therapeutic possibilities to break the triangle are discussed.
Abstract
The alteration of epigenetic modifications often causes cancer onset and development. In a similar way, aberrant alternative splicing may result in oncogenic products. These issues have often been individually reviewed, but there is a growing body of evidence for the interconnection of both causes of cancer. Actually, aberrant splicing may result from abnormal epigenetic signalization and epigenetic factors may be altered by alternative splicing. In this way, the interrelation between epigenetic marks and alternative splicing form the base of a triangle, while cancer may be placed at the vertex. The present review centers on the interconnections at the triangle base, i.e., between alternative splicing and epigenetic modifications, which may result in neoplastic transformations. The effects of different epigenetic factors, including DNA and histone modifications, the binding of non-coding RNAs and the alterations of chromatin organization on alternative splicing resulting in cancer are first considered. Other less-frequently considered questions, such as the epigenetic regulation of the splicing machinery, the aberrant splicing of epigenetic writers, readers and erasers, etc., are next reviewed in their connection with cancer. The knowledge of the above-mentioned relationships has allowed increasing the collection of biomarkers potentially useful as cancer diagnostic and/or prognostic tools. Finally, taking into account on one hand that epigenetic changes are reversible, and some epigenetic drugs already exist and, on the other hand, that drugs intended for reversing aberrations in alternative splicing, therapeutic possibilities for breaking the mentioned cancer-related triangle are discussed.
