Edited by: Rosanna Asselta, Humanitas University, Italy
Reviewed by: Dario Balestra, University of Ferrara, Italy; Mauricio Fernando Budini, University of Chile, Chile
†These authors have contributed equally to this work
This article was submitted to RNA, a section of the journal Frontiers in Genetics
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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, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.
The genome is the complete set of DNA that contains all the information necessary for the development and survival of an organism. In humans, the genome is contained in 23 chromosome pairs, comprising approximately 21,000 protein-coding genes and slightly over 3 billion DNA base pairs in total. Although the human genome was sequenced approximately fifteen years ago, there is still disagreement regarding the number of genes due to inconsistencies in the available databases (
Alternative splicing (AS), the phenomenon by which a single precursor (pre-) messenger RNA (mRNA) can generate alternative mRNAs to yield proteins with related or different functions, expands the protein information encoded by the genome. Global transcriptome analyses have estimated that 95–100% of multiexon genes undergo AS (
Alternative splicing in eukaryotes has considerable impacts on a variety of biological pathways; therefore, it is not surprising that AS is a highly orchestrated process involving multiple protein–protein and protein–RNA interactions. The spliceosome, the multiprotein complex that performs the splicing reaction, is composed of a core of five uridine-rich small nuclear RNAs (snRNAs; termed U1, U2, U4, U5, and U6) and 200 other proteins. The spliceosome assembles on pre-mRNA to remove noncoding introns through two sequential transesterification reactions: the branching and exon ligation steps. Detailing all the steps of the AS process is beyond the scope of this review, and comprehensive reviews on the molecular choreography of pre-mRNA splicing have been published elsewhere (
The major forms of AS include exon skipping, alternative 3′ and 5′ splice site (SS) usage, intron retention, and mutual exon exclusion (
Types of AS. In the graphs, exons are represented by boxes, and introns are represented by lines. Dashed lines indicate AS events. The five main types of AS are illustrated: exon skipping
Defects in core spliceosome components,
Defective control in pre-mRNA splicing leads to human diseases. Diagram showing how defects in
Recent advances in the development of novel technologies and tools, such as next-generation sequencing and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing technology, have greatly expanded the available information regarding RNA missplicing events and their association with diseases. These technologies have demonstrated the presence of many naturally occurring genetic variants that affect AS and lead to phenotypic variability and disease susceptibility among humans (
Mutations that occur in genes encoding fundamental components of the splicing machinery have been described in many splicing-related diseases. However, the frequency of these mutations is low, probably because their effects are incompatible with life. Disease-associated mutations that occur within introns lead to intron retention or to exon skipping upstream or downstream of the mutated SSs without affecting the coding sequence. In contrast, exonic mutations may or may not affect the coding sequence depending on the type of mutation (silent
With regard to cancer, genomic studies have identified frequent and recurrent mutations in genes that code for pre-mRNA splicing factors in both hematological malignancies (e.g., myelodysplastic syndrome [MDS], acute myeloid leukemia, and chronic lymphocytic leukemia) (
Recent advances in the treatment of some diseases have led to improvements in patient prognosis and life expectancy. For example, spinal muscular atrophy (SMA) type 1, which is considered to be most serious at an early age, can currently be treated with Zolgensma®. Zolgensma® is a new gene therapy-based drug approved by the United States Food and Drug Administration (FDA) that improves the quality of life and life expectancy of infants with SMA type 1. Although this treatment can cure this deadly inherited disease, the Swiss multinational corporation Novartis AG has established a sale price of 2.1 million dollars for a single intravenous administration. This drug is by far the most expensive pharmacological treatment in existence today.
Identification of splicing mutations has significantly advanced our understanding of how splicing dysregulation contributes to disease pathogenesis and of how splicing, a key pre-mRNA processing event, can be targeted for therapeutic applications. In
Designing effective therapeutic strategies to overcome the consequences of aberrant splicing events on disease states remains a major challenge. Gene therapy has emerged as a promising pharmacotherapeutic option for patients with diseases of genetic origin. Hence, targeting of aberrant RNA splicing is a logical approach for directly correcting disease-associated splicing alterations without affecting the genome. Other approaches, such as targeting splicing reactions to disrupt the expression of disease-related proteins or targeting exon junctions mutated mRNA to disrupt protein coding, can be used to reframe and rescue protein expression (
Several strategies have been designed to manipulate the splicing process, including spliceosome-mediated RNA
Schematic representation of different splicing-targeting strategies for gene modification.
Antisense oligonucleotides strategies use short synthetic single-stranded DNA molecules that are complementary to a specific pre-mRNA sequence to alter the splicing process. ASO binding to a target pre-mRNA in the nucleus sterically blocks the recruitment of
ASOs can be designed to target (i) the SS, thereby redirecting splicing to an adjacent site; (ii) auxiliary sequences (enhancers or silencer elements) within the immature transcript, thereby modifying the outcome of the splicing reaction (by blocking or promoting splicing); and (iii) RNA bases, thereby stabilizing/destabilizing regulatory structures and modifying the splicing outcome (
Splicing-related ASOs that act according to the first two mechanisms mentioned above by promoting or redirecting splicing are also called splice-switching oligonucleotides (SSOs). These short, 15- to 30-nucleotide-long sequences sterically block important motifs in pre-mRNA (i.e., SSs and/or regulatory sequences) to prevent RNA–RNA base-pairing or protein–RNA binding interactions between spliceosome components and pre-mRNA without promoting degradation of the RNA transcript while altering splicing outcomes (
The clinical application of this technology has resulted in the commercialization of VitraveneTM (fomivirsen), which, in 1998, became the first ASO approved by the FDA for the treatment of AIDS-related cytomegalovirus retinitis; MacugenTM (pegaptanib), approved by the FDA in 2004 for the treatment of neovascular age-related macular degeneration; and KynamroTM (mipomersen), approved by the FDA in 2013 for the treatment of homozygous familial hypercholesterolemia. These products have been withdrawn from the market for commercial reasons owing to an overall small patient population and competing alternative drugs, such as statins in the case of familial hypercholesterolemia (
In December 2016, the FDA approved SpinrazaTM (nusinersen), the first drug approved for the treatment of SMA the first drug approved for the treatment of SMA (
Investigational ASOs for Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and transthyretin (TTR) amyloidosis, such as RG6042, tofersen and inotersen, respectively, are currently in phase III clinical trials. RG6042 (previously known as IONIS-HTTRx) reduces the concentration of mutant huntingtin (HTT) levels in the cerebrospinal fluid of patients with HD without causing serious adverse events (
The development of the ASO milasen is an example of how cutting-edge medicine can be used with great speed for patient-customized treatment of a rare and fatal neurodegenerative disease (ceroid lipofuscinosis 7, CLN7, a form of Batten’s disease). Researchers at Boston Children’s Hospital identified a novel mutation in a 6-year-old girl, designed and produced an ASO, and obtained FDA approval for its clinical deployment in less than one year (
ASOs are also applicable to cancer treatment. For instance, Dewaele et al., used ASO-mediated exon skipping to decrease the expression of
Antisense oligonucleotides have been validated as therapeutic agents; however, because of the high cost associated with these products, improvements must be made to prevent health insurance companies from denying patients access. For instance, Exondys 51TM is priced at $300,000 per patient per year even though the efficacy of this drug is controversial.
Bifunctional oligonucleotides are ectopic modulators of AS used to control the patterns of splicing of specific genes. In brief, these oligonucleotides contain two parts: (i) an antisense portion targeting a specific sequence and (ii) a nonhybridizing tail or effector domain that recruits acting factors (targeted oligonucleotide enhancer of splicing [TOES] or targeted oligonucleotide silencer of splicing [TOSS]) (
In 1998, another related technology emerged with the discovery of the siRNA pathway, which can be used to silence the expression of genes (
These siRNA approaches can be used to target aberrant splicing isoforms for therapeutic applications (
In October 2016, the development of revusiran, an siRNA designed for the treatment of ATTR amyloidosis, was suspended after a randomized double-blind placebo-controlled phase III trial of its efficacy and safety, ENDEAVOUR, demonstrated that the siRNA caused greater mortality than a placebo (18 of 206 enrolled patients). However, in August 2018, the FDA approved OnpattroTM (patisiran), the first siRNA-based drug for the treatment of hereditary TTR-mediated amyloidosis (hATTR) (
Small-molecule compounds can also be used to modulate RNA expression. Some molecules are capable of binding specific three-dimensional RNA structures, thereby preventing their translation or function. Furthermore, these compounds can also modify splicing factor activity (by affecting posttranslational modifications of splicing factors) or directly alter splicing events. Compared with oligonucleotide-based therapeutics, these compounds are easier to deliver to target sites and normally have lower toxicity profiles. However, small-molecule compounds frequently act through unknown mechanisms, resulting in a lack of information, and have less target specificity than other therapeutic formulations, thus potentially exhibiting more nonspecific and off-target effects. While oligonucleotides (ASOs, TOSS, TOES and siRNAs) can very specifically and efficiently modulate particular RNA targets upon complementary recognition of the RNA sequences, small molecules can recognize specific three-dimensional structures and overcome known oligonucleotide drawbacks, e.g., poor pharmacological properties.
Some small molecules have already been approved for use in clinical practice for applications other than splicing defect correction. For example, digoxin and other prescribed cardiotonic steroids, routinely used in the treatment of heart failure, have been described as modulators of AS (
Some small-molecule splicing modulators have been evaluated in clinical trials for the treatment of solid tumors and leukemia. For instance, E7107 (pladienolide B) is a splicing modulator whose preferential cytotoxicity is positively influenced by some antiapoptotic
Recently, the Massachusetts-based pharmaceutical company Skyhawk Therapeutics invested $100 million in the development of small-molecule compounds capable of correcting misspliced exon skipping related to cancer and neurological diseases. Novartis and Roche have also independently developed two different splicing-modulating compounds for the treatment of SMA: branaplam and risdiplam, respectively. Both molecules enhance exon 7 inclusion to increase the levels of functional SMN protein. Branaplam, also known as LMI070, is an orally available drug that was designed by Novartis using a high-throughput phenotypic screening approach with approximately 1.4 million compounds. Currently, this molecule is in a phase II clinical trial that is expected to be completed in July 2020.
Risdiplam (
All these examples demonstrate the applicability of small molecules for splicing event modulation and suggest that these molecules are useful as complements and alternatives to oligonucleotides.
Spliceosome-mediated RNA trans-splicing is a gene-reprogramming system based on the
Depending on the targeted region of the pre-mRNA, SMaRT can be divided into (i) 5′-
Exon-specific U1 snRNAs (ExSpe U1s) are modified U1 snRNAs complementary to intronic regions downstream of the 5′ SS that can be used to eliminate the skipping of some exons caused by different mutations (
Other modified versions of spliceosomal snRNAs have also been tested for their usefulness in restoration of base pairing to the mutated SS. For example, combined treatment with mutation-adapted U1 and U6 snRNAs has been used to correct mutation-induced splice defects in exon 5 of the
The modified snRNA approach is based on engineered variants of small coding genes and has various advantages. Its main advantages are the possible exploitation of virtually any viral vector and the fact that, based on its molecular mechanisms, it does not alter the physiological expression of the target gene.
Zinc finger proteins are powerful and widely studied tools for efficient establishment of targeted genetic modifications (
Schematic representation of genome editing approaches. The three gene editing systems described in the main text are illustrated: ZFPs
Zinc finger nucleases were the first endonucleases designed for genome editing. In one such application, the association of ZF domains with the DNA cleavage domain of the restriction protein
Zinc finger nucleases can be used as therapeutic tools to correct genetic mutations associated with splicing-related diseases. The main advantage of ZFNs is that the correction of mutations is permanent; thus, continuous administration is not needed. For instance, Ousterout et al., applied ZFNs for permanent deletion of exon 51 of the dystrophin gene for the treatment of DMD (
The current and completed clinical trials of ZFN therapies (13 in total
These genome editing tools are chimeric nucleases engineered by fusion of the DNA-binding domain of the bacterial protein TALE with the catalytic domain of the restriction endonuclease
Fang et al. used TALENs
The development of CRISPR/Cas9 technology is considered one of the most important breakthroughs of the last decade. This nucleic acid immune system was first discovered in bacteria and archaea more than thirty years ago (
The CRISPR/Cas9 gene editing system requires administration of two main elements into a cell: (i) a small molecule of RNA (guide RNA [gRNA]) with a sequence complementary to that of the target mutation intended for editing and (ii) an endonuclease CRISPR-associated system (Cas) that allows the cleavage of the specific sequence under the guidance of gRNA binding (
Several studies have used CRISPR/Cas9 to correct splicing-related defects. Yuan et al. used a CRISPR-guided cytidine deaminase to successfully correct mutations associated with splicing-related diseases. These researchers used this tool to restore the expression and function of the protein dystrophin in DMD patients (
Despite the targeting specificity of Cas9, off-target DNA cleavage activity can occur. In addition, many recent reports have suggested that CRISPR/Cas9 might unintentionally generate alternatively spliced products, large genomic deletions, translocations and inversions; this is a matter of concern that should be further evaluated prior to the clinical application of this technology (
A modification of the CRISPR/Cas9 system termed the base editing system (BEs) has expanded the arsenal of tools for genome modification. Unlike CRISPR, the BEs can introduce precise base changes without introducing double-strand breaks (DSBs) and, in the case of HDR, without the requirement of a template donor (
Some of the strategies presented in this section are feasible treatment options for several diseases. Although the potential of nucleic acids (DNA or RNA) as drugs immediately became obvious decades ago, the actual development of nucleic acid-based medicines has faced major and evident hurdles. For instance, nucleic acids are highly susceptible to degradation by endogenous nucleases. Some of these nucleic acids, such as short oligonucleotides in their native form, have a very short half-life, even before they are filtered out through the kidneys. Large DNA/RNA constructs with highly negative charges cannot cross the vascular endothelium, dense extracellular matrix and cell and nuclear membranes to reach their intracellular DNA, pre-mRNA or mRNA targets. Moreover, off-target effects of many DNA/RNA therapeutic tools can lead to devastating adverse reactions. Finally, some of these tools can be immunogenic. Although chemical modifications can improve pharmacokinetic and pharmacodynamic properties, the ability of these promising therapeutic tools to efficiently deliver DNA/RNA in order to modify sufficient numbers of cells for therapeutic benefits is still the limiting factor for the translation of preclinical models to standard clinical care. This ongoing challenge, which is considered the Achilles heel of gene therapy (
In recent decades, various vectors and tools have been developed for gene therapy. In addition, the advent of new gene editing therapies, such as CRISPR/Cas9, has sparked investigation into appropriate gene delivery systems, including viral vector and transposon-based vector systems. Nanostructures are nanoscale-sized particles capable of transfecting cells and releasing cargoes such as small molecules, DNA, RNA and peptides to exert pharmacological effects. These nonviral vectors have received considerable attention due to their advantages compared to viral systems, which have been the most common choices for gene delivery. Several good reviews have extensively explained the differences between these types of vectors (
Several formulations based on nanoparticles have demonstrated sustained expression of transported cargoes and long-term achievement of biological effects (
Nanoparticles exist in different forms and can be divided into different classes based on their compositions and properties: polymeric nanoparticles, liposomes, lipid nanoparticles, and inorganic nanoparticles (
Schematic representation of different types of nanoparticles.
Polymeric nanoparticles are based on a polymeric matrix. The most frequently used polymers are poly-lactide-coglycolide (PLGA), polyhydroxyalkanoates (PHAs) and CDs (CDs) (
Polyhydroxyalkanoates are polyesters produced by microorganisms through, for example, bacterial fermentation of sugars or lipids. Because they are biodegradable and biocompatible polymers, PHAs have been used as bioimplant materials for medical and therapeutic applications for more than thirty years (reviewed in
CDs are cyclic oligosaccharides extensively used in pharmaceutical and biomedical applications. These molecules can be divided into 3 groups: α-CDs (6 subunits of glucose), β-CDs (7 subunits of glucose) and γ-CDs (8 subunits of glucose). CDs are biocompatible products approved by the FDA that are currently present in marketed formulations (
Liposomes were some of the first nanostructures to be developed for drug delivery. Liposomes were developed in the 1960s and are currently present in marketed formulations such as Doxyl®, Myocet® and Caelix®. At present, no liposome-based marketed formulations for gene delivery exist. Liposomes are nanoscale particles with a lipid bilayer composition that forms a spherical structure inside an aqueous compartment. In aqueous solution, liposomes form colloidal dispersions. The main components of liposomes are phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), PS, phosphatidylinositol (PI) and phosphatidyl glycerol (PG), and cholesterol, which can be incorporated into the phospholipid membrane to increase liposome stability (
Many published studies have shown the efficacy of liposomes in delivering RNA or DNA into cells. For example, Dorrani et al. developed a liposome formulation with DOTAP and sodium cholate (NaChol) as edge-activators that is capable of efficiently delivering siRNA through skin layers after topical administration, demonstrating that liposomes are good candidates for the treatment of skin diseases such as melanoma (
Lipid nanocarriers are some of the most promising nonviral tools for gene therapy. Currently, the only medicine approved by the FDA and European Medicines Agency (EMA) that uses nanostructures to deliver RNA is Onpattro® (mentioned in section “Small Interfering RNAs (siRNAs)”); Onpattro® is a lipid nanoparticle-based drug product that transports patisiran, an siRNA molecule for the treatment of TTR amyloidosis (
Lipid nanoparticles were developed to address important drawbacks of other lipid-based systems, such as instability and the necessity for use of surfactants and other toxic substances; to increase loading capacity; and to resolve other problems related to manufacturing and scale-up processes (
Solid lipid nanoparticles have been developed for gene delivery since 2000. The structure of SLNs can help to protect the drug or RNA/DNA against external agents and can enable modified and/or targeted release (
Nanostructured lipid carriers were first developed several years after SLNs. In contrast to SLNs, which involve solid lipids, NLCs involve liquid lipids; the use of liquid lipids increases the stability and drug loading capacity of the nanoparticles (
Inorganic nanoparticles include nanostructures that are manufactured using inorganic materials, such as gold, silicon and iron oxide; carbon materials; layered double hydroxide (LDH); or calcium phosphate (
Oxide nanoparticles can be classified into two important groups: silicon oxide nanoparticles and iron nanoparticles (
Iron nanoparticles can be made of different materials, such as magnetite (Fe3O4) and maghemite (Fe2O3). The intrinsic magnetic properties of these nanoparticles can be used for gene or drug delivery. For example, an external magnetic field can be used to guide the nanoparticles to a specific zone of the body for drug release. Furthermore, a magnetic field can be applied to a cell culture dish to enhance cell transfection by magnetic nanoparticles in a procedure known as magnetofection (
In 1993, the Nobel Prize in Physiology and Medicine was awarded to Phillip Sharp and Richard Roberts for their discovery of adenoviral RNA splicing (
All authors contributed to the writing and revision of the manuscript and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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