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Chronic kidney disease (CKD) is a global public health issue that places an increasing burden on the healthcare systems of both the developed and developing countries. CKD is a progressive and irreversible condition, affecting approximately 10% of the population worldwide. Patients that have progressed to end-stage renal disease (ESRD) require expensive renal replacement therapy, i.e., dialysis or kidney transplantation. Current CKD therapy largely relies on the use of angiotensin-converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs). However, these treatments by no means halt the progression of CKD to ESRD. Therefore, the development of new therapies is urgently needed. Antisense oligonucleotide (ASO) has recently attracted considerable interest as a drug development platform. Thus far, eight ASO-based drugs have been granted approval by the US Food and Drug Administration for the treatment of various diseases. Herein, we review the ASOs developed for the identification of CKD-relevant genes and/or the simultaneous development of the ASOs as potential therapeutics towards treating CKD.

The nucleotide monomer containing the 1-phenyl-1,2,3-triazole group attached to the 5-position of 2′-O-methyluridine is hereby presented together with a derivative further substituted with a p-sulfonamide group on the phenyl ring. Both were conveniently synthesised, and synergistic effect of the modifications were demonstrated when introduced into oligonucleotides and hybridised to complementary RNA. The combination of stacking of the phenyltriazoles and the conformational steering from the 2′-OMe group gave thermally very stable duplexes. Exon skipping in the distrophin transcript using 20-mer 2′-OMePS sequences with two phenyltriazoles introduced in different positions with and without the sulfonamide demonstrated efficient exon skipping but at the same level as the 2′-OMePS reference ASO.

Novel 3′-[4-fluoroaryl-(1,2,3-triazol-1-yl)]-3′-deoxythymidine analogues (7a-l) were developed by the Cu alkyne-azide cycloaddition (CuAAC) reaction. The obtained lead compounds were confirmed by using 1H NMR, 13C NMR, 2 D NMR, HRMS and their anticancer activities were screened against Huh-7 liver cancer cells and U87MG human glioblastoma cells. Among the synthesized fluorinated 1,2,3-triazolyl nucleosides, three compounds (7i, 7a-b) demonstrated promising anti-proliferative against Huh-7 and U87MG cell lines. Significantly, compound 7i has displayed remarkable promising anticancer activity with IC50 value in the micromole range (22.41–24.92 µM) and (18.12–21.36 µM) against Huh-7 cancer cells and U87MG glioblastoma cells, respectively.

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2′-O-methyl and 2′-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5–20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.

2′-O-Methyl (2′-OMe) antisense oligonucleotides (AOs) possessing a various number of 4-(trimethylammonio)butylsulfonyl or tosyl phosphoramidates (N+ and Ts-modifications, respectively) instead of a native phosphodiester linkage were designed to skip exon-23 in dystrophin pre-mRNA transcript in mdx mice myotubes. AOs bearing several zwitterionic N+ modifications in the sequence had remarkably increased thermal stability towards complementary mRNA in comparison with 2′-OMe-RNAs having negatively charged Ts and phosphorothioate (PS) linkages. However, only Ts-modified AOs exhibited a similar level of exon skipping in comparison with fully modified PS-containing 2′-OMe-RNA, whereas the exon skipping induced by N+ modified AOs was much lower with no exon-skipping detected for AOs having seven N+ modifications. The level of exon-skipping was improved once Ts and especially N+ moieties were used in combination with PS-modification, most likely through improved cellular and nuclear uptake of AOs. These results provide new insights on expanding the design of novel chemically modified AOs based on phosphate modifications.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2′OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2′OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2′OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.

Screening molecular libraries for ligands capable of binding proteins is widely used for hit identification in the early drug discovery process. Oligonucleotide libraries provide a very high diversity of compounds, while the combination of the polymerase chain reaction and DNA sequencing allow the identification of ligands in low copy numbers selected from such libraries. Ligand selection from oligonucleotide libraries requires mixing the library with the target followed by the physical separation of the ligand–target complexes from the unbound library. Cumulatively, the low abundance of ligands in the library and the low efficiency of available separation methods necessitate multiple consecutive rounds of partitioning. Multiple rounds of inefficient partitioning make the selection process ineffective and prone to failures. There are continuing efforts to develop a separation method capable of reliably generating a pure pool of ligands in a single round of partitioning; however, none of the proposed methods for single-round selection have been universally adopted. Our analysis revealed that the developers’ efforts are disconnected from each other and hindered by the lack of quantitative criteria of selection quality assessment. Here, we present a formalism that describes single-round selection mathematically and provides parameters for quantitative characterization of selection quality. We use this formalism to define a universal strategy for development and validation of single-round selection methods. Finally, we analyze the existing partitioning methods, the published single-round selection reports, and some pertinent practical considerations through the prism of this formalism. This formalism is not an experimental protocol but a framework for correct development of experimental protocols. While single-round selection is not a goal by itself and may not always suffice selection of good-quality ligands, our work will help developers of highly efficient selection

Synthetic antisense oligonucleotides (ASOs) have emerged as one of the most promising therapeutic approaches. So far, nine ASO drugs have received approval for clinical use, and four of them are based on splice-switching principles demonstrating the impact of ASO-mediated splice modulation. Notably, three among them (Exondys 51, Vyondys 53 and Viltepso) are based on phosphorodiamidate morpholino (PMO) chemistry whereas Spinraza is based on 2′-O-methoxyethyl phosphorothioate (2′-MOE PS) chemistry. Although systemic delivery of PMOs has displayed a good safety profile even at high doses, the 2′-O-methyl phosphorothioate modified (2′-OMe PS) ASO drug candidate (drisapersen) failed due to safety issues. The potency of 2′-modified RNA for splice-switching needs to be further improved by novel design strategies for broad applicability. Towards this goal, in this study, we evaluated the potential of incorporating DNA segments at appropriate sites in 2′-OMe PS and 2′-MOE PS ASOs to induce exon skipping. For this purpose, a four-nucleotide DNA segment was systematically incorporated into a 20-mer 2′-OMe PS and 2′-MOE PS ASO designed to skip exon 23 in mdx mouse myotubes in vitro. Our results demonstrated that 2′-modified RNA PS ASOs containing four or less PS DNA nucleotides at the 3′-end yielded improved exon 23 skipping efficacy in line with fully modified ASO controls. Based on these results, we firmly believe that the present study opens new avenues towards designing splice modulating ASOs with limited chemical modifications for enhanced safety and therapeutic efficacy.

Cancer is one of the leading causes of death worldwide. Conventional therapies, including surgery, radiation, and chemotherapy have achieved increased survival rates for many types of cancer over the past decades. However, cancer recurrence and/or metastasis to distant organs remain major challenges, resulting in a large, unmet clinical need. Oligonucleotide therapeutics, which include antisense oligonucleotides, small interfering RNAs, and aptamers, show promising clinical outcomes for disease indications such as Duchenne muscular dystrophy, familial amyloid neuropathies, and macular degeneration. While no approved oligonucleotide drug currently exists for any type of cancer, results obtained in preclinical studies and clinical trials are encouraging. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in oncology, review current clinical trials, and discuss associated challenges.

Lateral flow assay (LFA) has made a paradigm shift in the in vitro diagnosis field due to its rapid turnaround time, ease of operation and exceptional affordability. Currently used LFAs predominantly use antibodies. However, the high inter-batch variations, error margin and storage requirements of the conventional antibody-based LFAs significantly impede its applications. The recent progress in aptamer technology provides an opportunity to combine the potential of aptamer and LFA towards building a promising platform for highly efficient point-of-care device development. Over the past decades, different forms of aptamer-based LFAs have been introduced for broad applications ranging from disease diagnosis, agricultural industry to environmental sciences, especially for the detection of antibody-inaccessible small molecules such as toxins and heavy metals. But commercial aptamer-based LFAs are still not used widely compared with antibodies. In this work, by analysing the key issues of aptamer-based LFA design, including immobilization strategies, signalling methods, and target capturing approaches, we provide a comprehensive overview about aptamer-based LFA design strategies to facilitate researchers to develop optimised aptamer-based LFAs.

Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by >=30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.Copyright © 2021 The Author(s).

Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

Splice-modulating antisense therapy has shown tremendous potential in therapeutic development in recent years with four FDA-approved antisense drugs since 2016. However, an efficient and nontoxic antisense oligonucleotide (AO) delivery system still remains as a major obstacle in nucleic acid therapeutics field. Vitamin-E (α-tocopherol) is an essential dietary requirement for human body. This fat-soluble compound is one of the most important antioxidants which involves in numerous biological pathways. In this study, for the first time, we explored the scope of using α-tocopherol-conjugated bioresponsive AOs to induce splice modulation in mouse muscle myotubes in vitro. Our results showed that the bioresponsive construct efficiently internalized into the cell nucleus and induced exon 23 skipping in mdx mouse myotubes. Based on our exciting new results, we firmly believe that our findings could potentially benefit toward establishing a delivery approach to advance the field of splice-modulating AO therapy.

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2′-O-methyl phosphorothioate (2′-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2′-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2′-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from inefficient signaling and insufficient production of insulin. Conventional management of T2D has largely relied on small molecule-based oral hypoglycemic medicines, which do not halt the progression of the disease due to limited efficacy and induce adverse effects as well. To this end, antisense oligonucleotide has attracted immense attention in developing antidiabetic agents because of their ability to downregulate the expression of disease-causing genes at the RNA and protein level. To date, seven antisense agents have been approved by the United States Food and Drug Administration for therapies of a variety of human maladies, including genetic disorders. Herein, we provide a comprehensive review of antisense molecules developed for suppressing the causative genes believed to be responsible for insulin resistance and hyperglycemia toward preventing and treating T2D.

Brain cancer is one among the rare cancers with high mortality rate that affects both children and adults. The most aggressive form of primary brain tumor is glioblastoma. Secondary brain tumors most commonly metastasize from primary cancers of lung, breast, or melanoma. The five-year survival of primary and secondary brain tumors is 34% and 2.4%, respectively. Owing to poor prognosis, tumor heterogeneity, increased tumor relapse, and resistance to therapies, brain cancers have high mortality and poor survival rates compared to other cancers. Early diagnosis, effective targeted treatments, and improved prognosis have the potential to increase the survival rate of patients with primary and secondary brain malignancies. MicroRNAs (miRNAs) are short noncoding RNAs of approximately 18–22 nucleotides that play a significant role in the regulation of multiple genes. With growing interest in the development of miRNA-based therapeutics, it is crucial to understand the differential role of these miRNAs in the given cancer scenario. This review focuses on the differential expression of ten miRNAs (miR-145, miR-31, miR-451, miR-19a, miR-143, miR-125b, miR-328, miR-210, miR-146a, and miR-126) in glioblastoma and brain metastasis. These miRNAs are highly dysregulated in both primary and metastatic brain tumors, which necessitates a better understanding of their role in these cancers. In the context of the tumor microenvironment and the expression of different genes, these miRNAs possess both oncogenic and/or tumor-suppressive roles within the same cancer.

The hyperphosphorylation of the microtubule-associated protein tau (MAPT) has been implicated in various neurological diseases, including Alzheimer’s disease. It has been hypothesized that the reduction of MAPT would result in depolymerizing neurofibrillary tangles and could be a potential strategy for the treatment of Alzheimer’s disease and other tauopathies. In this study, we report the development of novel DNAzymes and splice-modulating antisense oligonucleotides (AOs) for the efficient inhibition of MAPT. We designed and synthesized a range of DNAzymes and 2ʹ-O-methyl (2’-OMe)-modified AOs on a phosphorothioate (PS) backbone targeting various exons across the MAPT gene transcript. Our results demonstrated that RNV563, an arm-loop-arm-type DNAzyme targeting exon 13, and an AO candidate AO4, targeting exon 4, efficiently downregulated MAPT RNA expression by 58% and 96%, respectively. In addition, AO4 also reduced the MAPT protein level by 74%. In line with our results, we believe that AO4 could be used as a potential therapeutic molecule for Alzheimer’s disease and other tauopathies.

Nucleic acid precipitation is important for virtually all molecular biology investigations. However, despite its crucial role, a systematic study of the influence factors of nucleic acid precipitation has not been reported. In the present work, via rational experimental design, key factors of nucleic acid precipitation, including the type of nucleic acid, temperature and time of incubation, speed and time of centrifugation, volume ratio of ethanol/isopropanol to nucleic acid solution, type of cation-containing salt solution and type of coprecipitator, were comprehensively evaluated in an attempt to maximize the efficiency of nucleic acid precipitation. Our results indicate that the optimal conditions of each influence factor of nucleic acid precipitation may vary in accordance with the chemistry, structure and length of nucleic acids.

A series of novel triazolylnucleosides has been efficiently synthesized (6a-h, 7a-b) by azide–alkyne 1,3-dipolar cycloaddition reaction and evaluated for antibacterial and anticancer activities. These nucleosides (6a-h) were screened against Staphylococcus aureus (ATCC 6538), gram-positive and Escherichia coli (ATCC 10536), Salmonella and Pseudomonas gram-negative bacterial strains. Among the series, three compounds (6c-e) exhibited significant levels of antibacterial activity against Staphylococcus aureus in reference to the standard drug, Levofloxacin. Triazolylnucleosides (6a-h) were also screened for anti-proliferative activities against cancer cell lines such as Huh-7 liver cancer cells. Three compounds (6e, 7a-b) have displayed promising anti-proliferative activity against Huh-7 and U87G cancer cells lines.

Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-42523-0, published online 15 April 2019

Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

An isolated or purified antisense oligomer targeted to a nucleic acid molecule encoding PTPN1 pre-mRNA, wherein the antisense oligomer inhibits the expression of PTP1B.

Amyloid beta-peptide is produced by the cleavage of amyloid precursor protein by two secretases, a β-secretase, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a γ-secretase. It has been hypothesised that partial inhibition of BACE1 in individuals with a high risk of developing Alzheimer’s disease may be beneficial in preventing cognitive decline. In this study, we report the development of a novel antisense oligonucleotide (AO) that could efficiently downregulate the BACE1 transcript and partially inhibit BACE1 protein. We designed and synthesised a range of 2’-OMethyl-modified antisense oligonucleotides with a phosphorothioate backbone across various exons of the BACE1 transcript, of which AO2, targeting exon 2, efficiently downregulated BACE1 RNA expression by 90%. The sequence of AO2 was later synthesised with a phosphorodiamidate morpholino chemistry, which was found to be not as efficient at downregulating BACE1 expression as the 2’-OMethyl antisense oligonucleotides with a phosphorothioate backbone variant. AO2 also reduced BACE1 protein levels by 45%. In line with our results, we firmly believe that AO2 could be used as a potential preventative therapeutic strategy for Alzheimer’s disease

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2 '-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2 '-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5 ' region of exon 51. These results indicated that locked nucleic acid/2 '-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

Low-density lipoprotein receptor (LDL-R) is a cell surface receptor protein expressed in a variety of solid cancers including lung, colon, breast, brain and liver, therefore opens up opportunities to deliver lysosome sensitive anti-cancer agents, especially synthetic nucleic acid-based therapeutic mole- cules. In this study, we focused on developing novel nucleic acid molecules specific to LDL-R. For this purpose, we performed in vitro selection procedure via SELEX methodologies using mammalian cell- expressed human recombinant LDL-R protein as a target. After ten rounds of selections, we identified a novel DNA oligonucleotide aptamer, RNV-L7, that can bind specifically to LDL-R protein with high affinity and specificity (Kd = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R over-expressed human cancer cells including Huh-7 liver can- cer cells and MDA-MB-231 breast cancer cells with a binding affinity of ∼200 nM. Furthermore, we evaluated the functional potential of the developed LDL-R aptamer RNV-L7 by conjugating with a pre- viously reported miR-21 targeting DNAzyme for inhibiting miR-21 expression. The results showed that the miR-21 DNAzyme-RNV-L7 aptamer chimera efficiently reduced the expression of miR-21 in Huh- 7 liver cancer cells. As currently there are no reports on LDL-R aptamer development, we believe that RNV-L7 could be beneficial towards the development of targeted cancer therapeutics.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the regulation of gene expression. Previous reports showed an over-expression of miRNA-21 (miR-21) in various cancer cells, and its up-regulation is closely related to cancer initiation, proliferation and metastasis. In this work, we envisioned the development of novel antimiRzymes (anti-miRNA-DNAzyme) that are capable of selectively targeting and cleaving miR-21 and inhibit its expression in cancer cells using the DNAzyme technique. For this purpose, we have designed different antimiRzyme candidates by systematically targeting different regions of miR-21. Our results demonstrated that RNV541, a potential arm-loop-arm type antimiRzyme, was very efficient (90%) to suppress miR-21 expression in U87MG malignant glioblastoma cell line at 200 nM concentration. In addition, RNV541 also inhibited miR-21 expression (50%) in MDA-MB-231 breast cancer cell line. For targeted delivery, we conjugated RNV541 with a transferrin receptor (TfR) targeting aptamer for TfR-mediated cancer cell delivery. As expected, the developed chimeric structure efficiently delivered the antimiRzyme RNV541 into TfR positive glioblastoma cells. TfR aptamer-RNV541 chimeric construct showed 52% inhibition of miR-21 expression in U87MG glioblastoma cells at 2000 nM concentration, without using any transfection reagents, making it a highly desirable strategy to tackle miR-21 over-expressed malignant cancers. Although these are in vitro based observations, based on our results, we firmly believe that our findings could be beneficial towards the development of targeted cancer therapeutics where conventional therapies face several challenges.

Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2′-Fluoro (2′-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2′-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2′-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2′-O-methyl (2′-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2′-F nucleotides induced efficient exon-23 skipping, with LNA/2′-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2′-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2′-OMe/2′-F chimeras. Overall, our findings certainly expand the scope of constructing 2′-F modified AOs in splice modulation by incorporating 2′-OMe and LNA modifications.

Epidermal growth factor receptor (EGFR) is associated with the progression of a wide range of cancers including breast, glioma, lung, and liver cancer. The observation that EGFR inhibition can limit the growth of EGFR positive cancers has led to the development of various EGFR inhibitors including monoclonal antibodies and small-molecule inhibitors. However, the reported toxicity and drug resistance greatly compromised the clinical outcome of such inhibitors. As a type of chemical antibodies, nucleic acid aptamer provides an opportunity to overcome the obstacles faced by current EGFR inhibitors. In this study, we have developed and investigated the therapeutic potential of a 27mer aptamer CL-4RNV616 containing 2′-O-Methyl RNA and DNA nucleotides. Our results showed that CL-4RNV616 not only displayed enhanced stability in human serum, but also effectively recognized and inhibited the proliferation of EGFR positive Huh-7 liver cancer, MDA-MB-231 breast cancer, and U87MG glioblastoma cells, with an IC50 value of 258.9 nM, 413.7 nM, and 567.9 nM, respectively. Furthermore, TUNEL apoptosis assay revealed that CL-4RNV616 efficiently induced apoptosis of cancer cells. In addition, clinical breast cancer biopsy-based immunostaining assay demonstrated that CL-4RNV616 had a comparable detection efficacy for EGFR positive breast cancer with commonly used commercial antibodies. Based on the results, we firmly believe that CL-4RNV616 could be useful in the development of targeted cancer therapeutics and diagnostics.

Aptamers, often termed as ‘chemical antibodies,’ are an emerging class of synthetic ligands for efficient target-specific molecular recognition. The objective of this book is to highlight recent advances and potential of aptamers in various disease conditions. . This book focuses on the applications of aptamers in targeted nanotherapy, detection, and in molecular imaging in various disease conditions such as cancer, neurological diseases and infectious diseases.

Cancer is one of the leading causes of death worldwide and conventional cancer therapies such as surgery, chemotherapy and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumour recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, Endoglin and Angiopoietins play important roles in regulating tumour development and metastasis, and serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognised by the US Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51 and Spinraza. Herein, we review the scope of antisense oligonucleotides that target angiogenic factors towards tackling solid cancers.

We have developed a novel functional nucleic acid aptamer to amyloid-β peptide 1–40 (Aβ1–40) and investigated its potential to detect Aβ peptide fragments in neuropathologically confirmed Alzheimer brain hippocampus tissues samples. Our results demonstrate that the aptamer candidate RNV95 could detect tetrameric/pentameric low-molecular-weight Aβ aggregates in autopsy hippocampal tissue from two neuropathologically confirmed Alzheimer disease cases. Although these are preliminary observations, detailed investigations are under way. This is the first demonstration of aptamer-Aβ binding in Alzheimer brain tissues.

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated w

We report a novel multifunctional hyperbranched polymer based on polyethylene glycol (PEG) as a nanomedicine platform that facilitates longitudinal and quantitative 89Zr-PET imaging, enhancing knowledge of nanomaterial biodistribution and pharmacokinetics/pharmacodynamics both in vivo and ex vivo. Anti-VEGF-A DNA aptamer functionalization increased tumour accumulation by >2-fold in a breast cancer model.

Aptamers are short single-stranded nucleic acid sequences capable of binding to target molecules in a way similar to antibodies. Due to various advantages such as prolonged shelf life, low batch to batch variation, low/no immunogenicity, freedom to incorporate chemical modification for enhanced stability and targeting capacity, aptamers quickly found their potential in diverse applications ranging from therapy, drug delivery, diagnosis, and functional genomics to bio-sensing. Aptamers are generated by a process called SELEX. However, the current overall success rate of SELEX is far from being satisfactory, and still presents a major obstacle for aptamer-based research and application. The need for an efficient selection strategy consisting of defined procedures to deal with a wide variety of targets is significantly important. In this work, by analyzing key aspects of SELEX including initial library design, target preparation, PCR optimization, and single strand DNA separation, we provide a comprehensive analysis of individual steps to facilitate researchers intending to develop personalized protocols to address many of the obstacles in SELEX. In addition, this review provides suggestions and opinions for future aptamer development procedures to address the concerns on key SELEX steps, and post-SELEX modifications.

Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.

Despite advancements in the development of high generation cationic-dendrimer systems for delivery of nucleic acid-based therapeutics, commercially available chemical agents suffer from major drawbacks such as cytotoxicity while being laborious and costly to synthesize. To overcome the aforementioned limitations, low-generation cationic peptide asymmetric dendrimers with side arm lipid (cholic and decanoic acid) conjugation were designed, synthesized and systematically screened for their ability to self-assemble into micelles using dynamic light scattering. Cytotoxicity profiling revealed that our entire asymmetric peptide dendrimer library when trialled alone, or as asymmetric dendrimer micelle-nucleic acid complexes, were non-cytotoxic across a broad concentration range. Further, the delivery efficiency of asymmetric peptide dendrimers in H-4-II-E (rat hepatoma), H2K (mdx mouse myoblast), and DAOY (human medulloblastoma) cells demonstrated that cholic acid-conjugated asymmetric dendrimers possess far superior delivery efficiency when compared to the commercial standards, Lipofectamine 2000 or Lipofectin®.

Duchenne muscular dystrophy (DMD) is a muscle wasting, invariably fatal genetic disease caused protein truncating mutations, commonly frame‐shifting deletions of one or more exons in the dystrophin gene that abolish functional dystrophin expression. Induced exon‐skipping therapy using splice‐switching antisense oligonucleotides (AO) has been explored in the treatment of DMD and is currently the only treatment that has been shown to unequivocally slow progression of the disease over several years. In this study, we explored novel nucleic acid chemistries such as anhydrohexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA) and Twisted Intercalating Nucleic Acids (TINA) and evaluating their exon‐skipping efficacy in vitro in the H2K mdx mouse cell line. We have designed, synthesised and tested the AOs to induce exon 23 skipping in Dmd pre‐mRNA. We also characterized critical properties of the AOs such as melting temperature with complementary RNA, stability against enzymatic activity, and cytotoxicity by WST‐1 assays. Overall, our results showed that all modified‐AOs efficiently induced exon 23 skipping in H2K cells in vitro, showed high nuclease resistance and no obvious toxicity. In addition, HNA, CeNA and ANA‐modified AOs also limited the unfavourable dual exon 22/23 skipping product. In conclusion, our study highlights the importance of exploring novel antisense chemistries in improving the scope of exon‐skipping therapy, and further systematic investigation of locked nucleic acid (LNA)‐modified AO‐based exon‐skipping is currently underway.

Antisense oligonucleotides (AOs) have gained significant interest in recent years towards the development of therapeutics against several diseases. AOs can be used to regulate the function of RNAs by skipping or retaining a specific exon inmessenger RNAs (mRNAs) during the RNA splicing process within the nucleus. To improve the pharmacodynamics of AOs, chemical modifications are normally used. Towards this, a number of nucleic acid analogues have been developed in recent years.On September 19th 2016, Eteplirsen, an AO with Phosphorodiamidate morpholino (PMO) chemistry targeting DMD exon‐51 has been conditionally approved by the US FDA for the treatment of Duchenne muscular dystrophy (DMD). We have investigated the potential of various chemically‐modified exon‐skipping AOs to induce exon‐23 skipping in DMD as model towards improving exon‐skipping efficacy. Very recently, we developed a novel analogue of PMO called morpholino nucleic acid (MNA) as the current PMO is not compatible with standard oligonucleotide chemistries, and large‐scale production is difficult making PMO very expensive. We investigated the potential of MNA to induce exon‐23 skipping in DMD, and found that the MNA‐modified AO efficiently induced exon‐23 skipping in mdx mice myotubes. We also investigated the potential of various other chemistries such as anhydrohexitol nucleic acid (HNA), althreitol nucleic acid (ANA), cyclohexenyl nucleic acid (CeNA), ortho‐twisted intercalating nucleic acids (oTINA) and locked nucleic acids (LNA) to induce exon‐23 skipping in DMD, and observed that these modifications can also yield efficient exon‐23 skipping.

Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2′-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2′-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2′-OMe-based oligos, by further reducing the length of AOs.

The design of inhibitors for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) is one of the most successful approaches for the treatment of HIV infections. Among the HIV-1 RT inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a prominent drug class, which includes nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine approved for clinical use. However, the efficiency of many of these drugs has been undermined by drug-resistant variants of HIV-1 RT, and it therefore becomes inevitable to design novel drugs to cope with resistance. Here, we discuss various drug design strategies, which include traditional medicinal chemistry, computational chemistry, and chemical biology approaches. In particular, computational modeling approaches, including machine learning, empirical descriptors-based, force-field, ab initio, and hybrid quantum mechanics/molecular mechanics-based methods are discussed in detail. We foresee that these methods will have a major impact on efforts to guide the design and discovery of the next generation of NNRTIs that combat RT multidrug resistance.

Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have been explored for exon-skipping applications. Herein, for the first time, we have investigated the potential of nucleobase-modified antisense oligonucleotides to induce exon-skipping. For this purpose, we have synthesised 5-(phenyltriazol)-2′-deoxyuridine-modified 2′-O-methyl mixmer antisense oligonucleotides, and evaluated their efficacy to induce exon-23 skipping in H-2Kb-tsA58 (H2K) mdx mouse myotubes as a model system. Our results showed that the phenyltriazol base-modified AOs successfully induced efficient exon-skipping in a DMD transcript. Our findings open up the exploration of novel base-modified antisense oligonucleotides for exon-skipping applications.

Nucleic acid-based technologies have received significant interest in recent years as novel theranostic strategies for various diseases. The approval by the United States Food and Drug Administration ( FDA) of Nusinersen, an antisense oligonucleotide drug, for the treatment of spinal muscular dystrophy highlights the potential of nucleic acids to treat neurological diseases, including Alzheimer's disease (AD). AD is a devastating neurodegenerative disease characterized by progressive impairment of cognitive function and behavior. It is the most common form of dementia; it affects more than 20% of people over 65 years of age and leads to death 7-15 years after diagnosis. Intervention with novel agents addressing the underlying molecular causes is critical. Here we provide a comprehensive review on recent developments in nucleic acid-based theranostic strategies to diagnose and treat AD.

Serinol nucleic acid (SNA) is a novel nucleic acid analogue that can form highly stable heteroduplexes with complementary DNA and RNA sequences. Structurally, SNA is a close mimic to peptide nucleic acid (PNA) which is widely used in diagnostic and therapeutic applications. SNA chemistry is relatively new, and so far the scope of SNA has only been explored in improving the efficacy of small interfering RNA and for developing a highly sensitive molecular beacon for diagnostic applications. In this study, we investigated the potential of SNA-modified antisense oligonucleotide (AO) in parallel to PNA-oligo for splice-modulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20-mer SNA and PNA antisense oligonucleotide (AO) designed to induce exon-23 skipping in the mouse dystrophin gene transcript. Our results demonstrated that the SNA AO induced exon-23 skipping at all tested concentrations, whereas the corresponding PNA AO failed to induce any exon-23 skipping upon 24 hours of transfection using Lipofectin transfection reagent. Our results further expands the potential of SNA oligonucleotides in therapeutic applications.

Twice as apt: Nucleic acid aptamers with high binding affinity, specificity, epitope coverage and nuclease resistance were developed by using libraries containing oligonucleotides in which two bases in the pyrimidine nucleotide had been modified.

Integrin alpha-4 (ITGA4) is a validated therapeutic target for multiple sclerosis (MS) and Natalizumab, an antibody targeting ITGA4 is currently approved for treating MS. However, there are severe side effects related to this therapy. In this study, we report the development of a novel DNAzyme that can efficiently cleave the ITGA4 transcript. We designed a range of DNAzyme candidates across various exons of ITGA4. RNV143, a 30mer arm-loop-arm type DNAzyme efficiently cleaved 84% of the ITGA4 mRNA in human primary fibroblasts. RNV143 was then systematically modified by increasing the arm lengths on both sides of the DNAzymes by one, two and three nucleotides each, and incorporating chemical modifications such as inverted-dT, phosphorothioate backbone and LNA-nucleotides. Increasing the arm length of DNAzyme RNV143 did not improve the efficiency however, an inverted-dT modification provided the most resistance to 3′ → 5′ exonuclease compared to other modifications tested. Our results show that RNV143A could be a potential therapeutic nucleic acid drug molecule towards the treatment for MS.

The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.

Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

In this study, we synthesised a morpholino nucleoside-uridine (MNA-U) phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO) sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2′-O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2′-OMePS) to the corresponding fully modified 2′-O-methyl RNA AO (2′-OMePS) as a control. Our results showed that the MNA/2′-OMePS efficiently induced exon 23 skipping. As expected, the 2′-OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides.