Interestingly, these researchers observed significant enrichment of epigenetic mark variants that may influence transcriptional activation and AS. In a comparative analysis of genome-wide association study GWAS data, many of the observed variants were found to be associated with various human diseases, particularly schizophrenia Takata et al.
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 versus missense or nonsense and can also alter the splicing pattern. Thus, mutations in introns or exons may disrupt RNA secondary structure or disrupt or create de novo cryptic SSs or de novo splicing silencers and enhancers, leading to dysregulation of AS. Mutations or quantitative changes in proteins with regulatory functions during the splicing process can also lead to aberrant splicing, affecting many RNA transcripts at the same time Havens et al.
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. These findings suggest a potential relationship between certain spliceosome gene mutations and carcinogenesis. Although the underlying mechanisms and contributions of splicing factors in cancer pathogenesis have not been elucidated, and although more work is needed to understand the splicing alterations observed in cancer cells, these data identify novel opportunities for development of splicing-based cancer therapies.
Recent advances in the treatment of some diseases have led to improvements in patient prognosis and life expectancy. Although this treatment can cure this deadly inherited disease, the Swiss multinational corporation Novartis AG has established a sale price of 2. 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 Supplementary Table 1 , we provide a list of the most frequent splicing-related human diseases that could be targeted for gene therapy. To learn more, readers are directed to several comprehensive reviews covering human diseases caused by RNA missplicing that have been published elsewhere Cieply and Carstens, ; Daguenet et al.
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 Havens et al. Several strategies have been designed to manipulate the splicing process, including spliceosome-mediated RNA trans -splicing SMaRT and the use of antisense oligonucleotides ASOs , bifunctional oligonucleotides, small-molecule compounds, and modified snRNAs Figure 3.
All of these approaches have been used to correct the effects of RNA misprocessing. Figure 3. Schematic representation of different splicing-targeting strategies for gene modification. 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.
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. Antisense oligonucleotides strategies use short synthetic single-stranded DNA molecules that are complementary to a specific pre-mRNA sequence to alter the splicing process. 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, to nucleotide-long sequences sterically block important motifs in pre-mRNA i. The nucleotides of an SSO are chemically modified e. Chemical modifications of ASOs are also crucial because they stabilize the ASOs in vivo and improve their cellular uptake, release and binding affinity for their targeted RNA sequences; unmodified oligonucleotides are highly susceptible to degradation by circulating nucleases and are excreted by the kidneys.
The clinical application of this technology has resulted in the commercialization of Vitravene TM fomivirsen , which, in , became the first ASO approved by the FDA for the treatment of AIDS-related cytomegalovirus retinitis; Macugen TM pegaptanib , approved by the FDA in for the treatment of neovascular age-related macular degeneration; and Kynamro TM mipomersen , approved by the FDA in 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 Sharma and Watts, The first published report on the use of ASOs as a splicing-targeting therapeutic tool was published by Dominski and Kole in Since then, many ASO strategies have been designed to modify splicing for the treatment of several diseases, and some of them are currently in clinical trials.
Exondys 51 TM belongs to the third generation of phosphorodiamidate morpholino ASOs and is specifically indicated for patients who have a confirmed mutation of the dystrophin gene amenable to exon 51 skipping.
Recently, Vyondys 53 TM golodirsen , a DMD drug that is highly similar to eteplirsen except that involves exon 53 skipping rather than exon 51 skipping, was denied by the FDA in August because of the risk of infections related to intravenous infusion ports and renal toxicity seen in preclinical studies.
A similar decision was reached in January for the promising drug Kyndrisa TM drisapersen. This drug was intended for the treatment of patients with DMD amenable to exon 51 skipping but failed to demonstrate substantial effectiveness. These products were in mid-stage trials for specific forms of the muscle-wasting disease. Notably, because ASOs generally do not cross the blood—brain barrier, repeated intrathecal nusinersen delivery is required.
This requirement is highly disadvantageous and makes administration challenging, especially for infants Verma, ASOs are also applicable to cancer treatment. For instance, Dewaele et al. Similarly, Hong et al. Ross et al. 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. 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] Brosseau et al.
Dikson et al. Similarly, bifunctional TOES, whose tail of enhancer sequences recruits activating proteins such as positively acting SR proteins, has been used to increase the splicing of refractory exon 7 in SMN2 in fibroblasts derived from patients with SMA Skordis et al. This approach is mechanistically different than ASO approaches, although both can be used for stimulating the inclusion of exon 7 in SMA.
In , another related technology emerged with the discovery of the siRNA pathway, which can be used to silence the expression of genes Fire et al. This discovery revolutionized the way scientists study gene function and offered an innovative strategy for the treatment of diseases, particularly those of genetic origin, as demonstrated for the first time by Elbashir et al.
These RNA types differ in structure, biological roles, associated effector proteins and origins Dana et al. Physiologically, in cells, siRNAs help to maintain genomic integrity by preventing the action of foreign nucleic acids, including those of viruses, transposons and retrotransposons and transgenes, while miRNAs act as posttranscriptional endogenous gene regulators Meister and Tuschl, These siRNA approaches can be used to target aberrant splicing isoforms for therapeutic applications Sune-Pou et al.
Such targeting approaches have been used for diseases such as Ullrich congenital muscular dystrophy UCMD Bolduc et al. In the context of cancer, it has been observed that the occurrence of specific splice variants is increased during tumorigenesis and that the splicing regulatory machinery is abnormal in many malignant cells Hayes et al.
Bolduc et al. These siRNAs resulted in specific knockdown of the mutant allele and increased the abundance and quality of collagen VI matrix production Bolduc et al. Similarly, Ryther et al. Finally, Hayes et al. Moreover, the sensitivity of tumor cells to chemotherapeutic agents such as gemcitabine and cisplatin increases upon treatment with this siRNA Hayes et al. Hence, siRNA-based technology has shown promising therapeutic results and the possibility of translation into clinical use.
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.
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 Stoilov et al. The histone deacetylase sodium butyrate, which is known to upregulate the expression of splicing factors, has been demonstrated to increase CFTR transcript levels, leading to activation of CFTR channels and restoration of their function in CFTR-derived epithelial cells Nissim-Rafinia and Kerem, Some small-molecule splicing modulators have been evaluated in clinical trials for the treatment of solid tumors and leukemia.
Furthermore, Aird et al. Notably, E was the first compound of a new class of anticancer agents targeting the spliceosome. Specifically, E interacts with subunit 1 of SF3b to block the normal splicing of oncogenes. Unfortunately, the development of E was suspended after phase I clinical trials due to an unacceptable profile of adverse events. Recently, Carabet et al. Thus, small molecules that selectively inhibit hnRNP A1-RNA interactions can be designed for the treatment of tumors expressing cancer-specific alternatively spliced proteins.
Similarly, highly specific inhibitors of the RNA helicase Brr2, which is an essential component of the spliceosome, have been designed for therapeutic purposes Iwatani-Yoshihara et al.
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 LMI, is an orally available drug that was designed by Novartis using a high-throughput phenotypic screening approach with approximately 1.
Currently, this molecule is in a phase II clinical trial that is expected to be completed in July Risdiplam Ratni et al. This drug is a splicing modulator that increases exon 7 inclusion in the SMN2 gene, thereby increasing the levels of SMN protein throughout the organism. Furthermore, this drug is being studied for use in patients of all age ranges with SMA types 1, 2, and 3. 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 trans -splicing process that can be used for therapeutic applications. The trans -splicing methodology is designed to correct aberrant mRNAs by replacing the entire coding sequence upstream or downstream of a target SS.
The first two components are present in cells, while the third must be provided exogenously. The results have shown that this technology can successfully reprogram gene expression and offers promising gene therapy applications. However, trans -splicing approaches require the use of vectors for the delivery of the PTM into cells. Therefore, selection of a good delivery vector is critical for future treatment approaches based on this technology Wally et al.
SMaRT offers multiple advantages as a gene therapy tool; however, it needs to be better understood and optimized in order to increase its overall efficiency. ExSpe U1s have been tested in different models and have shown potential for use in therapeutic applications Dal Mas et al.
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 BBS1 gene Schmid et al.
Changes in the target sequence can be introduced to convert this snRNA into an antisense tool capable of blocking splicing signals and inducing exon skipping or inclusion Brun et al. 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 Cristea et al.
Figure 4. Schematic representation of genome editing approaches. Zinc finger nucleases were the first endonucleases designed for genome editing.
To this end, two different ZFNs must recognize adjacent sequences separated by a spacer sequence where the break will be located. After this step, DNA repair pathways such as nonhomologous end joining NHEJ or homologous recombination HR with a codelivered exogenous DNA template lead to the establishment of a modified sequence in which the targeted mutation is corrected Cristea et al. Unfortunately, the use of ZFNs has some limitations, including high cost, off-target effects due to low specificity, and inappropriate interaction between domains.
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. The current and completed clinical trials of ZFN therapies 13 in total 1 , accessed in February focus on three major areas: cancer 1 clinical trial , blood disorders thalassemia, hemophilia B and sickle-cell disease: 3 clinical trials , infectious diseases human immunodeficiency virus [HIV]: 7 clinical trials and orphan diseases mucopolysaccharidosis: 2 clinical trials.
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 Fok I Schornack et al. Recognition of a specific DNA sequence is performed by the binding domain, which is composed of monomeric tandem repeats of 33—35 conserved amino acids; within this domain is a variable region known as the repeat variable diresidue RVD located at residues 12 and The RVD is responsible for binding to a specific nucleotide.
Fang et al. This nucleic acid immune system was first discovered in bacteria and archaea more than thirty years ago Ishino et al. Since these seminal reports, many researchers have contributed to the molecular understanding, technological development and medical applications of this gene editing system Lander, Although many Cas9 orthologs have been investigated, the most widely used is Cas9 from Streptococcus pyogenes.
Yuan et al. These researchers used this tool to restore the expression and function of the protein dystrophin in DMD patients Yuan et al. Foltz et al. These researchers were also able to differentiate each of these clones into retinal pigment epithelial cells with a nearly normal phenotype, highlighting the power and utility of this genome editing tool Foltz et al. Dastidar et al. These results support the use of this tool in developing new therapies for the treatment of DM1 Dastidar et al.
Kemaladewey et al. These observations, together with those described in a follow-up report by the same authors, validate the use of this gene editing technology as a therapeutic strategy for MDC1A Kemaladewi et al. Very recently, Stadtmauer et al.
The results of the trial demonstrated that it is feasible and safe to apply this technology for cancer immunotherapy Stadtmauer et al. Despite the targeting specificity of Cas9, off-target DNA cleavage activity can occur. Osborn et al. Recently, Song et al. The results showed improvements in liver histology in ABEs-treated mice, and the correction of the point mutation was confirmed by sequencing, indicating restoration from the diseased to the normal phenotype in vivo Song et al.
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. Finally, some of these tools can be immunogenic.
This ongoing challenge, which is considered the Achilles heel of gene therapy Somia and Verma, , is beginning to be overcome through the use of nanotechnologies.
These technologies use complexes of nucleic acids or encapsulate the nucleic acids in nonviral vectors, such as liposomes, lipids, and polymeric or inorganic nanoparticles, to enhance safe delivery to the target site. Next, we will focus on the application of nanotechnologies for gene delivery and discuss the advantages and problems associated with nanotechnology-based systems.
Addressing the problems will dismantle the barriers facing nucleic acid-based therapeutics. In recent decades, various vectors and tools have been developed for gene therapy.
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 Chira et al. The main advantage of nonviral gene delivery systems is their low immunogenicity, as high immunogenicity can impair viral transduction efficacy. Insertional mutagenesis is also a recognized safety concern associated with viral vectors intended for use in gene therapy Hacein-Bey-Abina et al. The major advantages of viral vectors include strong and prolonged transgene expression, broad cell tropism, and thorough understanding of viral gene function.
Compared with viral systems, nanoparticle-mediated nucleic acid delivery systems have the advantages of weak immunogenicity, lack of integration and absence of potential for viral recombination, all of which translate to improved safety Yin et al.
The development processes and manufacturing capacity for clinical-grade nanoparticles are also advantages of nanoparticle-based methods versus viral methods. Nanotechnologies are applicable to a large cohort of patients Paliwal et al. However, the transfection efficiency of nanoparticle-based systems is comparatively poor, and poor transfection efficiency is the main limitation for this and other nonviral methods.
For this reason, AAV vectors are the most commonly used vectors for nucleic-acid delivery. A good overview of the current status of the clinical translation of viral and nonviral systems for gene therapy has recently been published Kaemmerer, Several formulations based on nanoparticles have demonstrated sustained expression of transported cargoes and long-term achievement of biological effects Cohen et al.
Nanoparticles can also achieve successful tissue-specific delivery of biomolecules through different strategies. For example, incorporation of specific antibodies into the nanoparticle surface has enabled effective targeting of nanoparticles to the brain and lung endothelium Kolhar et al. Specific chemical components have also been incorporated into nanoparticles to increase delivery of biomolecules to targeted cells. 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 Figure 5.
Figure 5. Schematic representation of different types of nanoparticles. A Polymeric nanoparticles, B liposomes, C lipid nanoparticles, and D inorganic nanoparticles. Polymeric nanoparticles are based on a polymeric matrix. PLGA is a biodegradable and biocompatible polymer formed by units of lactic acid and glycolic acid. This excipient is approved by the FDA and has been extensively used to develop nanoparticles Zakharova et al. The grade of the polymer depends on the ratio between lactic acid and polyglycolic acid PGA , which can affect the final characteristics of the nanoparticle.
For example, polymers with higher glycolide contents have shorter deterioration times due to their more hydrophilic and amorphous characteristics. On the other hand, polymers with higher lactic content are more hydrophobic, thus exhibiting longer deterioration times Schliecker et al. Frequently, PLGA is mixed with other polymers to improve the characteristics of the resulting nanoparticles. For example, polyethylenimine PEI is commonly incorporated to improve the transfection efficiency of nanostructures Xie et al.
However, several studies have revealed that this polymer is cytotoxic, as elegantly discussed by Hunter more than a decade ago Hunter, Thus, the translation of PEI-based nanoparticles to clinical applications is limited. PEG is a nonionic biocompatible polymer coated onto the surfaces of nanoparticles that prevents recognition and destruction of these carriers by the mononuclear phagocyte system MPS , thereby increasing the plasma half-lives of the nanoparticles Mustafa et al.
Furthermore, PEGylation of nanoparticles improves their stability by reducing intermolecular aggregation and the accessibility of the target site Gref et al. PLGA-based nanoparticles can also be functionalized with ligands such as antibodies and Fab fragments to improve cellular targeting Kennedy et al. 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 Zhang et al.
Different types of PHAs can be used for nanoparticle formulation. PHA-based nanoparticles have been used to deliver biomolecules for anticancer Lu et al. A review discussing the use of PHA-based nanovehicles as therapeutic delivery carriers has been recently published Lin, CDs are cyclic oligosaccharides extensively used in pharmaceutical and biomedical applications. CDs are biocompatible products approved by the FDA that are currently present in marketed formulations Jambhekar and Breen, The cyclic structure of CDs results in a hydrophobic lumen and a hydrophilic surface.
This characteristic allows the use of CDs for multiple purposes, such as the vectorization of lipophilic drugs Fine-Shamir et al. Furthermore, CDs can penetrate cells and release their cargoes through, for example, pH-dependent mechanisms Tardy et al. CD-based nanoparticles have also been used for gene delivery. For example, Zuckerman et al.
Additionally, these researchers reported the functionalization of these nanoparticles with mannose or transferrin for enhanced nanoparticle uptake Zuckerman et al. Liposomes were some of the first nanostructures to be developed for drug delivery. 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 Bozzuto and Molinari, Other excipients can be used to improve the properties of liposomes or to endow them with new characteristics suitable for gene delivery.
In addition, polymers and carbohydrates, such as PEG and monosialoganglioside GM1 , can be incorporated into nanoparticle formulations to improve their in vivo half-lives and stability Daraee et al.
For example, Dorrani et al. Qiao et al. Lipid nanocarriers are some of the most promising nonviral tools for gene therapy. 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 Muller et al. Both types of nanoparticles use lipid excipients that are biocompatible and biodegradable.
These structures are highly attractive for clinical applications due to their simple and inexpensive manufacturing processes that do not require organic solvents and can be easily scaled up; their high stability; and their ability to be administered through different routes, such as the parenteral, pulmonary, oral and topical routes Uner and Yener, Solid lipid nanoparticles have been developed for gene delivery since SLNs are manufactured with solid lipid excipients.
The most common excipients include stearic acid, cholesterol derivatives e. To improve the efficacy of this type of vector, other excipients, such as protamine Limeres et al. There are many examples of the use of SLNs for gene therapy. For example, Apaolaza et al. Intravitreal administration of this formulation induced the expression of the protein retinoschisin in photoreceptors of Rs1h-deficient mice, leading to structural improvements in degenerated retinas Apaolaza et al.
Rassu et al. These researchers formulated nanoparticles with RVG-9R, a type of cell-penetrating peptide CPP that facilitated the transcellular pathway in neuronal cells. Furthermore, the researchers coated the nanoparticles with chitosan, which provided extra protection to the siRNA and increased the mucoadhesiveness of the particles, thereby increasing the residence time in the nasal cavity Rassu et al.
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 Uner and Yener, Other lipids used are canola stearin and myristyl myristate.
The other excipients are the same as those used for SLNs. A study by Taratula et al. The authors developed a multifunctional NLC-based system containing a drug paclitaxel or doxorubicin , two different types of siRNA, and a modified synthetic analog of luteinizing hormone-releasing hormone LHRH to increase specificity for local targeted delivery to lung tumors Taratula et al.
Similarly, Chen et al. Those authors tested the system in vitro and in vivo for efficient delivery to malignant glioblastoma cells for the treatment of malignant gliomatosis cerebri Chen et al. 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 Xu et al.
The easy surface functionalization, good target delivery and controlled release of these nanoparticles are their main advantages. Some of the most widely used inorganic nanostructures are gold nanoparticles AuNPs. This type of nanoparticle is used in the biomedical field for different applications, such as biodetection, biodiagnostics, and bioelectronic or therapeutic agent development.
One example of an AuNP-based gene delivery system was recently developed by Jia et al. The authors surface-conjugated AuNPs with thiol-modified antago-miR, an RNA antagonist to a potent promoter of proinflammatory type 1 macrophage polarization miR that plays an important role in diabetic cardiomyopathy.
In vivo administration of the AuNP complex resulted in the incorporation of nucleic acids into macrophages via phagocytosis and led to reduced inflammation, reduced apoptosis and restoration of cardiac function. Oxide nanoparticles can be classified into two important groups: silicon oxide nanoparticles and iron nanoparticles Xu et al. Mesoporous silica nanoparticles MSNs have been extensively investigated with regard to gene delivery. Furthermore, RNA and drugs can be loaded onto the same large pore-sized MSNs; thus, large pore-sized MSNs have the capacity to deliver two therapeutic agents at the same time.
Sun et al. The specific release of siRNA into the microtumor environment enables inhibition of MDR, and the subsequent release of doxorubicin enhances this effect Sun et al. Iron nanoparticles can be made of different materials, such as magnetite Fe 3 O 4 and maghemite Fe 2 O 3. 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 Estelrich et al. Despite the difficulties encountered in translating the magnetofection technique to clinical applications, advanced studies have demonstrated that iron nanoparticles can be applied for ex vivo delivery of chemically modified RNA cmRNA , opening the door to continuing studies on gene therapy applications Badieyan et al.
This discovery had notable consequences for elucidation of gene expression regulation and the evolution of eukaryotic cells. More than forty years after this seminal discovery, we have a deep understanding of the molecular mechanisms that control this important regulatory process, and we have recently begun to unravel the molecular links that connect faulty splicing with many human disorders.
This knowledge has enabled the design of innovative therapeutic strategies intended to correct splicing defects. Many tools based on nucleic acid gene repair have been tested with positive results, and many more tools warrant further development. The field is moving notably quickly, but we have attempted to provide a general overview of the main developments. However, as important as it is to decipher the mechanisms that govern the connections between missplicing and pathologies and to apply these findings in the clinic, research on the safe transport of therapeutic biomolecules into cells and to their targets is equally important.
Further development, characterization and testing of engineered technologies for targeted delivery and controlled release of DNA and RNA directly into cells with clinical applications are needed, as the demand for innovative nucleic acid delivery systems continues to grow. Nanoparticles possess considerable potential for use in the controlled delivery of therapeutic agents to specific target sites for splicing-based treatments.
Conducting related research is a challenging task, as basic scientists must interact and collaborate with nanotechnology experts. Funding opportunities should emphasize such collaboration as a way forward for grant support. 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.
Adli, M. Google Scholar. Aird, D. Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators. Alves, C. Further evidence of novel APOB mutations as a cause of familial hypercholesterolaemia.
Aneichyk, T. Dissecting the causal mechanism of X-linked dystonia-parkinsonism by integrating genome and transcriptome assembly. Anna, A. Splicing mutations in human genetic disorders: examples, detection, and confirmation. Apaolaza, P. The highly parallel and sensitive nature of microarrays make them ideal for monitoring gene expression on a tissue-specific, genome-wide level.
Microarray based methods for detecting splice variants provide a robust, scalable platform for high-throughput discovery of alternative gene splicing. A number of novel gene transcripts were detected using microarray based methods that were not detected by ESTs using computational methods. Another commonly used method for discovering of novel gene isoforms is RT-PCR followed by sequencing.
This is a powerful approach and can be effectively used for analyzing a small number of genes. However, it only provides only a limited view of the gene structure, is labor-intensive, and does not easily scale to thousands of genes or hundreds of tissues. Microarray based gene splicing detection poses some unique challenges in designing probes for isoforms that show a high degree of homology. In order to differentiate between these isoforms, a microarray that uses a combination of probes for exons and exon-exon junctions is used.
Exon skipping events or other deletions can be monitored by using junction probes. The results of different experiments that reported editing efficiencies for the same position and base editor were averaged together. To remove sgRNAs with potential off-targets, for each candidate sgRNA design, we scanned the genome for all sequences with at most two mismatches and calculated their off-target score [ 21 ]. We removed any sgRNA that has a top off-target score greater than An essential step during exon splicing is the recognition by the spliceosome machinery of the highly conserved sequences that define exons and introns.
More specifically, nearly every intron ends with a guanosine Fig. For this reason, we hypothesized that mutations that disrupt this guanosine within the splice acceptor of any given exon in genomic DNA would lead to exon skipping by preventing incorporation of the exon into mature transcripts.
We hypothesize that base editing of the highly conserved G asterisk leads to exon skipping. We selected an exon whose length is a multiple of 3 to ensure that exon skipping would not create a frameshift, which could lead to nonsense-mediated decay and complicate the detection of novel splicing events.
By gel electrophoresis, we observed that exon skipping is detectable for the first time 4 days after transfection, but the skipping frequency increases significantly on days 6, 8, and 10 Fig. Based on these data we chose to analyze all subsequent experiments 6 days after transfection. Single-base editing of splice acceptor consensus sequences enables programmable exon skipping. RT-PCR was used to detect exon skipping over a day time course.
Since the transfection efficiency in these cell lines is typically lower than that in T cells, we enriched for transfected cells prior to analysis, which revealed successful skipping of the targeted exon in all cell lines tested. In parallel, sgRNAs targeting the same exons were co-transfected with active SpCas9 to induce exon skipping. At this target, the exonic base was modified without modifying the intronic base in only 2.
Deep sequencing was performed in biological duplicates, and the results were combined. Cas9 can bind DNA even when the sgRNA is not perfectly matched, which can result in undesired modifications in the genome.
We found that 14 out of 18 Therapeutic exon skipping often requires inducing splicing of multiple exons simultaneously within the same transcript to recover a reading frame [ 17 ].
RT-PCR demonstrated that both sgRNAs induced skipping of the targeted exon and, when used together, induced skipping of both exons simultaneously. We estimate that these four base editors together enable targeting of , out of , inner exons in protein coding transcripts genome assembly version GRCh38 and GENCODE release 26 at the off-target score [ 21 ] cutoff of 10, where corresponds to perfect matching on targets Fig.
Only exons with maximum off-target score below 10 are considered. For example, it may enable the study of alternatively spliced genes whose various protein isoforms have distinct roles in tissue specification and development [ 23 ].
Furthermore, interrogating lncRNA by transcriptional silencing is complex, because their promoters are frequently multi-functional and regulate expression of multiple elements [ 25 ]. In addition to recovering the reading frame of mutant genes, CRISPR-SKIP allows for isoform-specific modulation that cannot be achieved by introducing premature stop codons through current gene-editing strategies [ 26 , 27 , 28 ].
Since the changes introduced by CRISPR-SKIP are hardwired in the genome after a single treatment, this technology is especially attractive as a potential therapeutic tool for a wide variety of human diseases. It is noteworthy that in our experiments we achieved statistically significant base editing at Detailed analysis of each of these exons revealed the presence of cryptic splice acceptor sites, which may have been activated when the native site was destroyed.
While the reason why base editors fail to induce skipping of some exons remains unknown, we anticipate that improved understanding of exon—intron architecture and their recognition by the spliceosome machinery will enable more efficient targeting in the future. Similarly, advancements in base-editing technologies will likely improve the rate of exon skipping as well as span the number of exons that can be effectively targeted. Interestingly, in the samples in which the target exon was successfully skipped, we observed some discrepancies between genomic DNA editing efficiency and the measured rate of exon skipping, which could be explained by the lower number of splice events that an exon-skipped transcript must undergo.
In fact, one of the major blocks during transcript elongation is the splicing junction [ 38 , 39 , 40 ], as demonstrated by the findings that splicing leads to transient polymerase pausing at the splice sites [ 41 ]. Only three of these mutations occurred in coding sequences, but it will be important for future applications of CRISPR-SKIP to mitigate off-target effects by using newer generations of base editors.
More specifically, target specificity can be increased through several recent additions to the base-editing toolkit, such as high fidelity base editors that have decreased affinity for genomic DNA and thus rely on longer sequences of sgRNA—DNA base complementarity for binding [ 42 ].
Another example is the base editor BE4-GAM, which, by decreasing indels introduced by Cas9 nickase, has been shown to reduce unintended mutations [ 43 ]. However, we have observed that the exon-skipping activity of BE4-GAM is lower than the activity of BE3 at some target sites Additional file 2 : Figure S7 ; therefore, it is important to test various base editors to identify a proper balance between activity and specificity. Our off-target analysis also supports that off-target effects are mostly derived from off-target Cas9 binding, indicating that high-fidelity base editors [ 42 ] may effectively decrease CRISPR-SKIP off-target mutations while preserving activity.
When the purpose of the experiment is to skip an exon already containing a mutation, the impact of this bystander mutation will likely be minimal. For applications in which this mutation is unacceptable, there are several alternative approaches to shift the editing window. When this is not an option, Cas9 variants that have been engineered specifically for editing within narrow windows can be used [ 10 ]. Finally, the linker connecting the Cas9 scaffold and the cytidine deaminase plays a critical role defining the cytidine that is modified as well as the modification rate [ 11 , 43 ].
Therefore, it is possible that optimization of the domain structure of base editors may prevent bystander mutations. In terms of specificity, however, it is important to note that the stochasticity of DSB repair, as well as the potential for translocations and other chromosomal aberrations that are not typically detected by current methods for analyzing off-target modifications, renders active Cas9 less predictable and potentially less safe than CRISPR-SKIP.
The results presented in this manuscript demonstrate that programmable exon skipping can be accomplished by disrupting splice acceptors using single-base editors. Given the current availability of various base editors that use different Cas9 scaffolds, we estimate that , out of , inner exons in protein coding transcripts can be targeted. We demonstrated that this method is multiplexable, is applicable to multiple cell lines of diverse species, and can achieve skipping rates as high as Our study also provides a webtool for rapidly identifying and designing potential target sites in the entire human genome.
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Nat Methods. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.
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