Publications by authors named "Rakesh N Veedu"

75 Publications

Oligonucleotides containing 2'-O-methyl-5-(1-phenyl-1,2,3-triazol-4-yl)uridines demonstrate increased affinity for RNA and induce exon-skipping in vitro.

Bioorg Med Chem 2021 Dec 12;55:116559. Epub 2021 Dec 12.

Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2021.116559DOI Listing
December 2021

Antisense Oligonucleotide-Mediated Splice Switching: Potential Therapeutic Approach for Cancer Mitigation.

Cancers (Basel) 2021 Nov 5;13(21). Epub 2021 Nov 5.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Murdoch, WA 6150, Australia.

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.
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http://dx.doi.org/10.3390/cancers13215555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583451PMC
November 2021

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization.

Chembiochem 2021 Dec 22;22(24):3437-3442. Epub 2021 Oct 22.

Chemistry Department, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.

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.
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http://dx.doi.org/10.1002/cbic.202100413DOI Listing
December 2021

Sequence-dependent inhibition of cGAS and TLR9 DNA sensing by 2'-O-methyl gapmer oligonucleotides.

Nucleic Acids Res 2021 06;49(11):6082-6099

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia.

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.
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http://dx.doi.org/10.1093/nar/gkab451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216285PMC
June 2021

Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis.

Theranostics 2021 5;11(11):5174-5196. Epub 2021 Mar 5.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150, Australia.

Lateral flow assay (LFA) has made a paradigm shift in the 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.
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http://dx.doi.org/10.7150/thno.56471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039946PMC
July 2021

Recent Advances in Oligonucleotide Therapeutics in Oncology.

Int J Mol Sci 2021 Mar 24;22(7). Epub 2021 Mar 24.

Department of Biochemistry, University of Otago, Dunedin 9016, New Zealand.

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.
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http://dx.doi.org/10.3390/ijms22073295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036554PMC
March 2021

How to Develop and Prove High-Efficiency Selection of Ligands from Oligonucleotide Libraries: A Universal Framework for Aptamers and DNA-Encoded Small-Molecule Ligands.

Anal Chem 2021 04 25;93(13):5343-5354. Epub 2021 Mar 25.

Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.

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 approaches to consolidate their efforts under an umbrella of universal quantitative criteria of method development and assessment.
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http://dx.doi.org/10.1021/acs.analchem.1c00601DOI Listing
April 2021

Antisense Oligonucleotides as Potential Therapeutics for Type 2 Diabetes.

Nucleic Acid Ther 2021 02 7;31(1):39-57. Epub 2020 Oct 7.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, Australia.

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.
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http://dx.doi.org/10.1089/nat.2020.0891DOI Listing
February 2021

Therapeutically Significant MicroRNAs in Primary and Metastatic Brain Malignancies.

Cancers (Basel) 2020 Sep 7;12(9). Epub 2020 Sep 7.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Murdoch, WA 6150, Australia.

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.
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http://dx.doi.org/10.3390/cancers12092534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564168PMC
September 2020

Novel Disulfide-Bridged Bioresponsive Antisense Oligonucleotide Induces Efficient Splice Modulation in Muscle Myotubes in Vitro.

ACS Omega 2020 Jul 16;5(29):18035-18039. Epub 2020 Jul 16.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, 90 South Street, Murdoch, Perth, Western Australia 6150, Australia.

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 . Our results showed that the bioresponsive construct efficiently internalized into the cell nucleus and induced exon 23 skipping in 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.
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http://dx.doi.org/10.1021/acsomega.0c01463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391367PMC
July 2020

Development of Novel Chemically-Modified Nucleic Acid Molecules for Efficient Inhibition of Human Gene Expression.

Genes (Basel) 2020 06 19;11(6). Epub 2020 Jun 19.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150, Australia.

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 2'-O-methyl (2'-OMe)-modified AOs on a phosphorothioate (PS) backbone targeting various exons across the 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 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.
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http://dx.doi.org/10.3390/genes11060667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349501PMC
June 2020

Author Correction: Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro.

Sci Rep 2020 Apr 16;10(1):6669. Epub 2020 Apr 16.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, 6150, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-63706-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162846PMC
April 2020

Alpha-l-Locked Nucleic Acid-Modified Antisense Oligonucleotides Induce Efficient Splice Modulation In Vitro.

Int J Mol Sci 2020 Mar 31;21(7). Epub 2020 Mar 31.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150 Australia.

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 exon-23 skipping in 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.
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http://dx.doi.org/10.3390/ijms21072434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177859PMC
March 2020

Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.

Theranostics 2020 19;10(8):3684-3707. Epub 2020 Feb 19.

School of Nursing, Zhengzhou University, Zhengzhou, 450001, China.

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.
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http://dx.doi.org/10.7150/thno.41580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069071PMC
May 2021

A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation.

Biotechniques 2020 04 18;68(4):191-199. Epub 2020 Feb 18.

Henan Key Laboratory of Ion-beam Bioengineering, School of Physics, Zhengzhou University, Zhengzhou 450000, China.

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.
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http://dx.doi.org/10.2144/btn-2019-0109DOI Listing
April 2020

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries.

Front Genet 2019 20;10:1249. Epub 2019 Dec 20.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA, Australia.

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 transcripts were synthesized as two different chemistries, 2'--methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and transcripts were analyzed for exon skipping. The ineffective 2'--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'--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.
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http://dx.doi.org/10.3389/fgene.2019.01249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951410PMC
December 2019

Development of a Novel DNA Oligonucleotide Targeting Low-Density Lipoprotein Receptor.

Mol Ther Nucleic Acids 2020 Mar 16;19:190-198. Epub 2019 Nov 16.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, Australia; Perron Institute for Neurological and Translational Science, Perth, WA 6009, Australia. Electronic address:

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, and therefore it opens up opportunities to deliver lysosome-sensitive anti-cancer agents, especially synthetic nucleic acid-based therapeutic molecules. 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 systematic evolution of ligands by exponential enrichment (SELEX) methodologies using mammalian cell-expressed human recombinant LDL-R protein as a target. After 10 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 (K = 19.6 nM). Furthermore, flow cytometry and fluorescence imaging assays demonstrated efficient binding to LDL-R overexpressed human cancer cells, including Huh-7 liver cancer 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 previously 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 think that RNV-L7 could be beneficial toward the development of targeted cancer therapeutics.
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http://dx.doi.org/10.1016/j.omtn.2019.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920325PMC
March 2020

The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth.

Int J Oncol 2019 Dec 4;55(6):1223-1236. Epub 2019 Oct 4.

Ghrelin Research Group, Translational Research Institute-Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland 4102, Australia.

Recent evidence suggests that numerous long non‑coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription‑quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA‑MB‑231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA‑MB‑231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.
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http://dx.doi.org/10.3892/ijo.2019.4891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831199PMC
December 2019

BACE1 Inhibition Using 2'-OMePS Steric Blocking Antisense Oligonucleotides.

Genes (Basel) 2019 09 12;10(9). Epub 2019 Sep 12.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150, Australia.

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 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 transcript, of which AO2, targeting exon 2, efficiently downregulated 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 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.
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http://dx.doi.org/10.3390/genes10090705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770983PMC
September 2019

Efficient Epidermal Growth Factor Receptor Targeting Oligonucleotide as a Potential Molecule for Targeted Cancer Therapy.

Int J Mol Sci 2019 Sep 22;20(19). Epub 2019 Sep 22.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth 6150, Australia.

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.
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http://dx.doi.org/10.3390/ijms20194700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801465PMC
September 2019

Development of Novel antimiRzymes for Targeted Inhibition of miR-21 Expression in Solid Cancer Cells.

Molecules 2019 Jul 7;24(13). Epub 2019 Jul 7.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, WA 6150, Australia.

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.
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http://dx.doi.org/10.3390/molecules24132489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651226PMC
July 2019

Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro.

Sci Rep 2019 04 15;9(1):6078. Epub 2019 Apr 15.

Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, 6150, Australia.

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.
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http://dx.doi.org/10.1038/s41598-019-42523-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465270PMC
April 2019

Antisense Oligonucleotides Targeting Angiogenic Factors as Potential Cancer Therapeutics.

Mol Ther Nucleic Acids 2019 Mar 20;14:142-157. Epub 2018 Nov 20.

Centre for Comparative Genomics, Murdoch University, Murdoch, WA 6150, Australia; Perron Institute for Neurological and Translational Science, Nedlands, WA 6009, Australia. Electronic address:

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 tumor recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, endoglin, and angiopoietins, play important roles in regulating tumor development and metastasis, and they 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 recognized by the U.S. 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 toward tackling solid cancers.
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http://dx.doi.org/10.1016/j.omtn.2018.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307321PMC
March 2019

Three decades of nucleic acid aptamer technologies: Lessons learned, progress and opportunities on aptamer development.

Biotechnol Adv 2019 Jan - Feb;37(1):28-50. Epub 2018 Nov 5.

Centre for Comparative Genomics, Murdoch University, Perth 6150, Australia; Perron Institute for Neurological and Translational Science, Perth 6009, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biotechadv.2018.11.001DOI Listing
July 2019

Radiolabeling of Nucleic Acid Aptamers for Highly Sensitive Disease-Specific Molecular Imaging.

Pharmaceuticals (Basel) 2018 Oct 15;11(4). Epub 2018 Oct 15.

Centre for Comparative Genomics, Murdoch University, Perth 6150, Australia.

Aptamers are short single-stranded DNA or RNA oligonucleotide ligand molecules with a unique three-dimensional shape, capable of binding to a defined molecular target with high affinity and specificity. Since their discovery, aptamers have been developed for various applications, including molecular imaging, particularly nuclear imaging that holds the highest potential for the clinical translation of aptamer-based molecular imaging probes. Their easy laboratory production without any batch-to-batch variations, their high stability, their small size with no immunogenicity and toxicity, and their flexibility to incorporate various functionalities without compromising the target binding affinity and specificity make aptamers an attractive class of targeted-imaging agents. Aptamer technology has been utilized in nuclear medicine imaging techniques, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET), as highly sensitive and accurate biomedical imaging modalities towards clinical diagnostic applications. However, for aptamer-targeted PET and SPECT imaging, conjugation of appropriate radionuclides to aptamers is crucial. This review summarizes various strategies to link the radionuclides to chemically modified aptamers to accomplish aptamer-targeted PET and SPECT imaging.
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http://dx.doi.org/10.3390/ph11040106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315947PMC
October 2018

Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides.

Molecules 2018 Oct 8;23(10). Epub 2018 Oct 8.

Centre for Comparative Genomics, Murdoch University, Perth, WA 6150, Australia.

Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.
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http://dx.doi.org/10.3390/molecules23102564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222501PMC
October 2018

Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro.

Chem Commun (Camb) 2018 May;54(36):4593-4596

Centre for Comparative Genomics, Murdoch University, Perth, 6150, Australia.

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.
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http://dx.doi.org/10.1039/c8cc02256aDOI Listing
May 2018

Self-assembling asymmetric peptide-dendrimer micelles - a platform for effective and versatile in vitro nucleic acid delivery.

Sci Rep 2018 03 19;8(1):4832. Epub 2018 Mar 19.

School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Brisbane, QLD, Australia.

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.
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http://dx.doi.org/10.1038/s41598-018-22902-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859181PMC
March 2018

Rational Design of Short Locked Nucleic Acid-Modified 2'-O-Methyl Antisense Oligonucleotides for Efficient Exon-Skipping In Vitro.

Mol Ther Nucleic Acids 2017 Dec 12;9:155-161. Epub 2017 Sep 12.

Centre for Comparative Genomics, Murdoch University, Perth, WA 6150, Australia; Perron Institute for Neurological and Translational Science, Perth, WA 6009, Australia. Electronic address:

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-2K-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.
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http://dx.doi.org/10.1016/j.omtn.2017.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633351PMC
December 2017

Development of Cell-Specific Aptamers: Recent Advances and Insight into the Selection Procedures.

Molecules 2017 Nov 27;22(12). Epub 2017 Nov 27.

Centre for comparative Genomics, Murdoch University, Perth 6150, Australia.

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.
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http://dx.doi.org/10.3390/molecules22122070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149766PMC
November 2017
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