For example, chimeric ASOs that fuse SR recruitment domains (RS peptide) to an antisense sequence that suppresses silencer motifs have been shown to increase exon 7 inclusion and SMN protein levels in patient fibroblasts; a single injection of the bifunctional ASO was able to induce SMN protein in the brain of neonatal mice and resulted in extended lifespan.65,69 This ASO design strategy, known as ESSENCE (exon-specific splicing enhancement by small chimeric effectors), was initially pursued in SMA therapeutics, but later studies showed that the RS peptide was not necessary to achieve a robust effect on SMN2 exon 7 inclusion. ASO is a catch-all term designing short single-stranded RNA molecules, with length ranges from seed-targeting 8-mer oligonucleotides to a fully complementary mature miRNAs [68], specifically inhibiting their function [69], and hence, acting as miRNA antagonists. Spinraza (nusinersen) was approved by the US FDA in December, 2016, for treatment of all types of SMA and all ages. ASO drugs are also being investigated to be used in combination with nanoparticles. Cell uptake is predominantly mediated by endocytosis. M. Kumar, ... J.I. Antisense oligonucleotide (ASOs) are small-sized single-stranded nucleic acids and offer some advantage over siRNAs in terms of targeting both nuclear and cytoplasmic located lncRNAs. in their work on transgenic mice found these intronic splicing suppressors to be located in intron 7 as tandem motifs, namely, hnRNP A1/A2. Genetic Disorders Affecting the Motor Neuron, Swaiman's Pediatric Neurology (Sixth Edition), Protein and Peptide Nanoparticles for Drug Delivery, Advances in Protein Chemistry and Structural Biology, Beltinger et al., 1995; Lebedeva & Stein, 2001, Arnedo, Irache, González Gaitano, Valgañón, & Espuelas, 2003; Arnedo, Irache, Merodio, & Espuelas Millán, 2004, Arnedo, Espuelas, & Irache, 2002; Arnedo et al., 2004, Spinal Muscular Atrophy Therapeutics Development, Huntingtonâs Disease and Other Polyglutamine Repeat Diseases, The Molecular and Cellular Basis of Neurodegenerative Diseases, Martinez, Wright, Lopez-Fraga, Jimenez, & Paneda, 2013, Boudreau et al., 2009; Kordasiewicz, Stanek, & Wancewicz, 2012, Skotte, Southwell, & Ostergaard, 2014; Stanek, Yang, & Angus, 2013. We use cookies to help provide and enhance our service and tailor content and ads. Splicing regulatory elements play an important role in determining which sites are recognized by the cellular splicing machinery. in their work on transgenic mice found these intronic splicing suppressors to be located in intron 7 as tandem motifs, namely hnRNP A1/A2. By continuing you agree to the use of cookies. Phase I/II clinical trials and two randomized, double-blind, sham procedure controlled phase III trials demonstrated the safety, tolerability and clinical efficacy of multiple doses of ASO (nusinersen), which is delivered intrathecally into the subarachnoid space of SMA type I and type II patients. As an ASO, IONIS-HTTRx binds to Htt RNA, promoting its destruction and decreasing the amount of Htt protein to cause toxicity. They are generally introduced in vitro by transfection and in vivo by viral transduction. Antisense oligonucleotides: We can use a method in which by designing some short-nucleotide sequences specific to the mRNA we wish to silence, to make a gene inactive. Based on their sequence homology, ASOs bind to their target RNA sequence inside the cells and bring about gene silencing. ASOs are single-stranded nucleic acids synthesized to be complementary to a target RNA sequence. ASO dosage reduction can be ânonallele specific,â meaning that both the disease allele and the normal allele mRNAs are targeted, or âallele-specific,â meaning that the disease allele is selectively targeted, usually because of a single nucleotide polymorphism that is in linkage with the disease allele. We use cookies to help provide and enhance our service and tailor content and ads. Preclinical studies of nusinersen in nonhuman primates demonstrated that a single intrathecal injection could result in the tissue concentrations necessary for significant improvements in exon 7 inclusion and those concentrations were maintained for several months.62 The drugâs long half-life means patients may only need an intrathecal injection every few months.73 In a phase 1 open-label study in SMA type II/III patients of ages 2â14 years, a single injection of nusinersen was well tolerated and appears to have correlated with increased HFMSE scores at the highest dosage.74 In June 2015, Ionis released new data from an ongoing open-label phase 2 study of nusinersen in type I infants, which showed the infants lived longer and achieved more motor milestones than what would normally be expected from the diseaseâs natural history.75 In addition, the drugâs mechanism of action was supported by data showing an increase in the amount of full-length SMN2 mRNA and SMN protein in brain and spinal cord tissues from autopsies of patients who received treatment. Another option overcoming the potential pharmacological caveat would be to use antisense oligonucleotides (ASO) targeting AhR or SMAD3 (Leclerc et al, 2021). Nanobody-functionalized nanoparticles showed 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells in comparison with PEGylated nanoparticles. In order to stabilize AS ONs against nucleolytic degradation, chemically modified nucleotides such as phosphorothioates, 2â²-O-methyl RNA, or locked nucleic acids are widely employed because they confer nuclease resistance. Copyright © 2021 Elsevier B.V. or its licensors or contributors. Additional modifications of the sugar at the 2′ position, increase RNA binding affinity and significantly increase potency, tissue half-life and prolong RNA inhibitory activity. Ito, S. Obika, in Comprehensive Medicinal Chemistry III, 2017, Phosphorothioated ASOs are believed to be distributed in vivo by multiple proteins. For the Supplementary Data which include background information and detailed discussion of the data that have provided the basis for the Guidelines see https:// demonstrated enhanced production of full-length SMN mRNA in fibroblasts from patients treated by ASOs. The backbone chemistry of an ASO can impact suitability for various delivery methods, the necessity for repeat dosing, and confer associated toxicitiesâ all of which have important implications for its therapeutic use. As discussed in earlier chapters, SMN2 differs from SMN1 by a C-to-T transition that occurs six nucleotides into exon 7 (there are other differences between the two sequences, but this is the most important functionally). The C-to-T transition creates an extended inhibitory context in exon 7 (also known as âExinctâ).63 Because exon 7 is otherwise weakly defined due to a weak 5â²-splice site, the C-to-T mutation in SMN2 is sufficient to tip the balance from its inclusion to exclusion. IONIS-HTTRx is the first therapy to enter clinical development that is designed to treat the underlying cause of this fatal disease and has been granted orphan drug designation by the FDA and the European Medicines Agency. S.S. Sunshine, ... K.S. This strategy, in addition to increasing the specificity of the drug, also reduces the side effects when compared with conventional chemotherapy. The substitution of one non-bridging oxygen with the more hydrophobic sulfur atom (phosphorothioate) increases both plasma stability and plasma protein binding and thus, ultimately, tissue bioavailability. Each patient will receive four doses of IONIS-HTTRx or placebo by an intrathecal (spinal) injection, with doses 4 weeks apart. This method is known as antisense oligonucleotides. Preclinical studies in rodents and nonhuman primates have successfully shown that α-Syn can be downregulated in PD-affected brain areas after direct application of oligonucleotide therapeutics including antisense oligonucleotides (ASO), small interfering RNAs (siRNA), and microRNAs (miRNA) [24,25,26,27,28,29,30,31]. The oligonucleotides composing an ASO are typically modified to optimize pharmaceutical properties such as cellular uptake, toxicology, pharmacokinetics, and target binding. Single-stranded antisense oligonucleotides (ASOs) interact with proteins on the surface of cells and enter the cytoplasm by endocytosis. Because of dramatic successes in preclinical trial work in mice, ASO therapy is being advanced into human patients. Successful release of the kinase inhibitor in the target cell and inhibition of proliferation were observed from targeted nanoparticles, whereas the nontargeted formulations had no antiproliferative effects on 14C cells. Several improvements have been made with ASOs including using locked nucleic acids for better stability, lesser off-target effects, free uptake by the cell, lesser cytotoxicity in nontarget cells, etc. Improved Cellular Delivery of Antisense Oligonucleotide for miRNA-21 Imaging In Vivo Using Cell-Penetrating Peptide-Based Nanoprobes. Tissue bioavailability (% of administered dose) is assisted by plasma protein binding that limits glomerular filtration and ultimate urinary excretion of oligonucleotides. Since a chimeric approach introduces difficulties in manufacturing the ASO and limits the ASOâs cellular permeability, bifunctional ASOs have not been tested extensively in mice and are not currently being developed clinically.62. The complexation of ASOs with albumin nanoparticles protects unmodified ASOs against enzymatic degradation and improves their nucleus accumulation and biological activity (Arnedo, Irache, González Gaitano, Valgañón, & Espuelas, 2003; Arnedo, Irache, Merodio, & Espuelas Millán, 2004). Regardless of backbone, any ASO designed for SMA therapy will likely need to be directly administered to the cerebrospinal fluid via intrathecal injection. They can also bind with Watson-Crick base-pairing complementarity to a specific mature mRNA in order to alter its function through various mechanisms, consequently reverting the natural miRNA effect on this gene target [70]. Significant reduction of PIK1 mRNA and protein expression was observed by ASOs-loaded trastuzumab-modified nanoparticles in BT474 breast cancer cells using real-time polymerase chain reaction (PCR) and Western blot analyses. Dosing mice with the same amount of monomer or homodimer ASO (10 mg kgâ 1 and twice a week) demonstrated that the homodimer exhibits much higher antisense potency than its monomer.78, Chiraz Atri, ... Dhafer Laouini, in AGO-Driven Non-Coding RNAs, 2019. Pharmacokinetic properties of oligonucleotides are largely driven by chemistry of the backbone and thus are sequence independent within a chemical class. Although albumin nanoparticles prepared by desolvation showed high ASO efficacy, the use of glutaraldehyde can lead to an inactivation of ASOs through chemical cross-linking due to its toxic entity. They are also able to alter the alternative RNA splicing and induce the target mRNA cleavage through RNase H [73, 74, 76â78], recruited in both the nucleus and the cytoplasm [79]. Once ASOs targeting ISS-N1 were shown to be remarkably effective at promoting exon 7 inclusion, the next strategic question was identifying which ASO backbone would be best used in a potential SMA therapy. Research studies with IONIS-HTTRx in animal models of HD have been promising (Skotte, Southwell, & Ostergaard, 2014; Stanek, Yang, & Angus, 2013). ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Published by Elsevier B.V. https://doi.org/10.1016/j.addr.2015.01.008. Hasan Kouchakzadeh, ... Seyed Abbas Shojaosadati, in Advances in Protein Chemistry and Structural Biology, 2015. Both size and charge for most oligonucleotides prevents distribution across the blood brain barrier. By continuing you agree to the use of cookies. (B) shRNAs consist of sense and antisense sequences separated by a loop sequence. While double-stranded ASOs use the RISC complex to degrade their mRNA target, single-stranded ASOs reduce gene expression by different mechanisms. However, modified single-strand oligonucleotides administered by intrathecal injection into the CSF distribute broadly in the CNS. However, one shortcoming of these approaches is their lack of mutant versus wild-type (WT) specificity. Systemic biodistribution is broad, and organs typically with highest concentrations are liver and kidney followed by bone marrow, adipocytes, and lymph nodes. Herschkowitz, in Progress in Molecular Biology and Translational Science, 2016. Antisense oligonucleotides (ASO) are synthetic single-stranded nucleic acid sequences that can be deployed with similar function as siRNAs, though bear ⦠Subsequently, they escape the endosome and enter the nucleus, where they bind to the SMN2 pre-mRNA and block an intronic splicing suppressor element located in intron 7. Once administered, the ASO is taken up into the cell via an as yet poorly defined endocytic mechanism and then enters the nucleus where it binds to its target pre-mRNA.62 Initially, ASOs were used to suppress gene expression either by blocking translation of an RNA target or promoting its degradation. Guangjie Yang, Yujun Zhao, Aidi Gong, Wenjie Miao, Lei Yan, Pei Nie*, and ; Zhenguang Wang* The association of ASOs with albumin carriers is simple and does not require the addition of any positive compounds (Arnedo, Espuelas, & Irache, 2002; Arnedo et al., 2004). SMA therapeutic ASOs are designed to modify the alterative splicing of SMN2 pre-mRNA so that exon 7 is included in the final processed transcript. SiRNA can increase the apoptosis of breast cancer cells by reducing the gene expression of SALL4 gene and Bcl-2; it can be used as a novel targeted therapy. Structure of siRNA and shRNA. Audrey S. Dickey, Albert R. La Spada, in The Molecular and Cellular Basis of Neurodegenerative Diseases, 2018. (A) siRNAs are short RNA duplexes with characteristic 2 nt 3â overhangs. Hua etâ¯al. Copyright © 2015 The Authors. The majority of intracellular oligonucleotide distribution following systemic or local administration occurs rapidly in just a few hours following administration and is facilitated by rapid endocytotic uptake mechanisms. They have successfully been employed to inhibit gene expression, modulate splicing of a precursor messenger RNA, or inactivate microRNAs. However, the widespread use of these molecules in clinic has been limited by two major obstacles, including their enzymatic instability and poor and inadequate intracellular delivery (Beltinger et al., 1995; Lebedeva & Stein, 2001). Two types of ASOs have been used to study gene function and in clinical trials: âmorpholinoâ ASOs that impair mRNA translation into protein, and âgapmerâ ASOs responsible for mRNA destruction through the recruitment of the RNase H enzyme that specifically hydrolyses RNA within the RNA:DNA duplex [71]. In either case, the production of protein product from the target mRNA is typically reduced by greater than one-half. Krieg et al. Antisense oligonucleotides (ASOs) have been developed that block an intronic splicing suppressor element, which in turn prevents skipping of exon 7 (Figure 8.8). Steinhauser et al. Analysis of SDHB RNA expression from cBioPortal database shows deletions as a major genetic aberration. Genta Incorporated was a biopharmaceutical company started in La Jolla, California, which discovered and developed innovative drugs for the treatment of patients with cancer.Founded in 1989 by a highly skilled entrepreneur, the company focused on a novel technology known as antisense, which targets gene products that are associated with the onset and progression of serious diseases. Further understanding of the intracellular trafficking of oligonucleotides may provide further enhancements in design and ultimate potency of antisense oligonucleotides in the future. Figure 8.8. AR downregulation by either siRNA (upper panel) or antisense oligonucleotides (lower panel) in LNCaP or LAPC4 cells decreased SDHA and SDHB subunit protein expression. Adapted from Addgene and Biosettia. Basil T. Darras, ... Darryl C. De Vivo, in Swaiman's Pediatric Neurology (Sixth Edition), 2017. Antisense oligonucleotides (ASOs) have been developed that block an intronic splicing suppressor element, which in turn prevents skipping of exon 7 (Burghes and McGovern, 2010). Chen, in Spinal Muscular Atrophy, 2017. After the last dose, patients will be followed for several weeks to monitor the safety and activity of IONIS-HTTRx. The pharmacokinetics and biodistribution of oligonucleotides has been predominantly published for antisense single-stranded DNA/RNA oligonucleotides (ASOs) and, to a lesser extent, double-stranded siRNA. Reduced by greater than one-half characteristic 2 nt 3â overhangs is undertaking a similar approach to the cerebrospinal via... Any ASO designed for SMA a loop sequence of mutant versus wild-type ( WT ) specificity nanoparticles... Mrna expression via siRNA or antisense oligonucleotides oligonucleotides ( ASOs ) interact with proteins on the targeted region the... 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