Antisense Applications Research Articles

Enhancing Antisense Oligonucleotide-Based Therapeutic Delivery with DG9, a Versatile Cell-Penetrating Peptide.

Antisense oligonucleotide-based (ASO) therapeutics have emerged as a promising strategy for the treatment of human disorders. Charge-neutral PMOs have promising biological and pharmacological properties for antisense applications. Despite their great potential, the efficient delivery of these therapeutic agents to target cells remains a major obstacle to their widespread use. Cellular uptake of naked PMO is poor. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular uptake and intracellular delivery of oligonucleotide-based drugs. Among these, the DG9 peptide has been identified as a versatile CPP with remarkable potential for enhancing the delivery of ASO-based therapeutics due to its unique structural features. Notably, in the context of phosphorodiamidate morpholino oligomers (PMOs), DG9 has shown promise in enhancing delivery while maintaining a favorable toxicity profile. A few studies have highlighted the potential of DG9-conjugated PMOs in DMD (Duchenne Muscular Dystrophy) and SMA (Spinal Muscular Atrophy), displaying significant exon skipping/inclusion and functional improvements in animal models. The article provides an overview of a detailed understanding of the challenges that ASOs face prior to reaching their targets and continued advances in methods to improve their delivery to target sites and cellular uptake, focusing on DG9, which aims to harness ASOs' full potential in precision medicine.

Exploring Nucleobase Modifications in Oligonucleotide Analogues for Use as Environmentally Responsive Fluorophores and Beyond.

Over the past two decades, it has become abundantly clear that nucleic acid biochemistry, especially with respect to RNA, is more convoluted and complex than previously appreciated. Indeed, the application and exploitation of nucleic acids beyond their predestined role as the medium for storage and transmission of genetic information to the treatment and study of diseases has been achieved. In other areas of endeavor, utilization of nucleic acids as a probe molecule requires that they possess a reporter group. The reporter group of choice is often a luminophore because fluorescence spectroscopy has emerged as an indispensable tool to probe the structural and functional properties of modified nucleic acids. The scope of this review spans research done in the Hudson lab at The University of Western Ontario and is focused on modified pyrimidine nucleobases and their applications as environmentally sensitive fluorophores, base discriminating fluorophores, and in service of antisense applications as well as tantalizing new results as G-quadruplex destabilizing agents. While this review is a focused personal account, particularly influential work of colleagues in the chemistry community will be highlighted. The intention is not to make a comprehensive review, citations to the existing excellent reviews are given, any omission of the wonderful and impactful work being done by others globally is not intentional. Thus, this review will briefly introduce the context of our work, summarize what has been accomplished and finish with the prospects of future developments.

Cytotoxic effect of combining two antisense oligonucleotides against telomerase rna component (hTR and mRNA of centromere protein B (CENP-B) in hepatocellular carcinoma cells.

Telomerase is a ribonucleoprotein enzyme that plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation and helping in the high rate proliferation of cancer cells. Our study is designed to evaluate the effect of using combined antisense oligonucleotides (ASOs) targeting (hTR) and mRNA of CENP-B on liver cancer cells. Compared with a single treatment, combination treatment with Locked Nucleic Acid (LNA) ASO (hTR) and (CENP-B) (6.25 nM from each) exhibit the maximum synergistic cytotoxic effect. hTR and CENP-B mRNA was abrogated while hTERT expression was disappeared. Caspase-3, Bax, and Bcl-2 were not detected, indicating caspase-independent cell death. A significant reduction in [Tumor necrosis factor (TNF-α) and Transforming growth factor (TGF-β)] coincides with elevation in Nitric oxide (NO) secretions was observed. Taken together; our data suggest that combination treatment with LNA ASO (hTR) and (CENP-B) could provide a promising strategy for cancer treatment by controlling many pathways concurrently. This might open a new prospective application of antisense in cancer therapy.

Abstract PO055: A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides

Abstract Background: Current cancer treatment tactics ought to be selective to carcinogenic cells and non-destructive to normal cells. Telomerase is a ribonucleoprotein enzyme which plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. While it is not found in somatic cells, its reactivation is obligatory in malignant cells to maintain the length of the telomere and prevent its senescence. Antisense-based strategies against human Telomerase RNA component (hTR) led to cancer cell death at some time after administering of antisense oligonucleotide (ASOs), and this lag time is one of the major drawbacks for this approach in treating cancer. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation. CENP-B is highly expressed in cancer cells helping in high rate proliferation. Materials and Methods: HepG2 cells were transfected with several concentrations (6.25, 12.5 and 25 nM) of LNA ASO against hTR and CENP-B mRNA either individually or in combination. MTT assay was used to assess the Hep-G2 cytotoxicity 24 and 48 hrs post-transfection. Telomerase activity, hTR, and CENP-B mRNA were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Effect of antisense treatment on Caspase-3, Bax, and Bcl-2 was also investigated by RT-PCR. Transforming growth factor (TGF-β), Tumor necrosis factor (TNF-α) and, Nitric oxide (NO) were estimated in supernatant collected 48 after transfection with ASOs by enzyme-linked immunosorbent assay (ELISA). Results: Our study conclusively demonstrates that using a single treatment with LNA ASO- hTR or CENP-B led to dose and time-dependent reduction in HepG2 cell viability. The combination of two LNA antisense (hTR + CENP-B) with different concentrations showed a synergistic cytotoxic effect. LNA ASO treatments (6.25 nM from each) exhibit the greatest synergistic cytotoxic effect. hTR and CENP-B mRNA in HepG2 cells were totally abrogated. Telomerase activity was disappeared. Expression of Caspase-3, Bax, and Bcl-2 was not detected after transfecting cells with LNA ASO hTR alone or in-combination with LNA ASO CENP-B indicates to caspase-independent cell death. A significant decrease in TGF-β and TNF-α and elevation in NO secretions was observed. Conclusion: LNA ASO hTR is potent in decreasing telomerase activity with a rapid anti-tumor effect with Caspase-independent cell death. This tumor inhibitory effect was magnified by ASO CENP-B indicating to the synergistic cytotoxic effect of oligonucleotides combination at certain concentrations. The rapid inhibitory effects of combined oligonucleotides on tumor growth open a new prospective application of antisense in cancer therapy. Citation Format: Ahmed Mohamed El-Desoky, Yasser Ali, Roba Talaat. A novel therapeutic approach for targeting HCC via combined two LNA-Gapmer antisense oligonucleotides [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO055.

Molecular dynamics simulations of acyclic analogs of nucleic acids for antisense inhibition

For antisense applications, oligonucleotides must be chemically modified to be resistant to endogenous nucleases. Until now, antisense oligonucleotide (ASO) analogs have been synthesized and then tested for their ability to duplex with a target nucleic acid, usually RNA. In this work, using molecular dynamics calculations simulations, we systematically tested a series of chemically modified analogs in which the 2-deoxyribose was substituted for by one or two methylene groups on each side of the phosphate backbone, producing four compounds, of which three were previously unknown. We used a 9-mer sequence of which the solution structure has been determined by NMR spectroscopy and tested the ability to form stable duplexes of these acyclic analogs to both DNA and RNA. In only one case out of eight, we unexpectedly found the formation of a stable duplex with complementary RNA. We also applied limitations on end fraying because of the terminal AT base pairs, in order to eliminate this as a factor in the comparative results. We consider this a predictive method to potentially identify target ASO analogs for synthesis and testing for antisense drug development.

Thioamide-Bridged Nucleic Acid (thioAmNA) Containing Thymine or 2-Thiothymine: Duplex-Forming Ability, Base Discrimination, and Enzymatic Stability.

Oligonucleotides containing bridged nucleic acids (BNAs) show high duplex-forming ability towards target single-stranded RNA, so many BNAs have been developed for antisense applications. Amide-bridged nucleic acids (AmNAs), which are BNA analogues bearing an amide bond at the bridge, exhibit high duplex-forming ability, enzymatic stability, and antisense activity; thus, the AmNA motif represents a promising BNA scaffold. The high enzymatic stability of the AmNA motif is presumably attributable to the bulky amide structure, because it inhibits the access of nucleases to the phosphodiester linkage. Here, to improve enzymatic stability further, we designed thioAmNAs: thioamide-bridged nucleotides that have a bulkier bridge structure than AmNA. The synthesis of thioAmNAs bearing either thymine (thioAmNA-T) or 2-thiothymine (thioAmNA-S2 T) bases was successful, and the obtained monomers were introduced into designed oligonucleotides without noticeable by-product generation. The thioAmNA-T- and thioAmNA-S2 T-modified oligonucleotides showed strong binding affinity toward complementary single-stranded RNA, with the thioAmNA-S2 T-modified oligonucleotide displaying excellent base-discrimination capability. Moreover, both thioAmNA-T and thioAmNA-S2 T endowed oligonucleotides with higher resistance to enzymatic degradation than AmNA-T. These results indicate that thioAmNAs are potentially useful chemical modifications for oligonucleotide-based therapeutics.

Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications.

Antisense oligonucleotides (ASOs) are becoming important drugs for hard to treat diseases. Modifications to their DNA backbones are essential to inhibit degradation in vivo, but they can reduce binding affinity to RNA targets. To address this problem we have combined the enzymatic resistance of carbamate (CBM) DNA backbone analogues with the thermodynamic stability conferred by locked nucleic acid sugars (LNA). Using a dinucleotide phosphoramidite strategy and automated solid phase synthesis, we have synthesised a set of oligonucleotides modified with multiple LNA-CBM units. The LNA sugars restore binding affinity to RNA targets, and in this respect LNA position with respect to the CBM linkage is important. Oligonucleotides containing carbamate flanked on its 5'and 3'-sides by LNA form stable duplexes with RNA and unstable duplexes with DNA, which is desirable for antisense applications. Carbamate-LNA modified oligonucleotides also show increased stability in the presence of snake venom and foetal bovine serum compared to LNA or CBM backbones alone.

Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs).

Dinucleoside phosphoramidites containing a triazole internucleotide linkage flanked by locked nucleic acid (LNA) were synthesized and incorporated into oligonucleotides (ONs). ONs bearing both LNA and triazole at multiple sites were obtained and their biophysical properties including enzymatic stability and binding affinity for RNA and DNA targets were studied. t-LNAs with four incorporations of a dinucleoside monomer having LNA on either side of the triazole linkage bind to their RNA target with significantly higher affinity and greater specificity than unmodified oligonucleotides, and are remarkably stable to nuclease degradation. A similar but reduced effect on enzymatic stability and binding affinity was noted for LNA only on the 3′-side of the triazole linkage. Thus, by combining unnatural triazole linkages and LNA in one unit (t-LNA), we produced a promising class of ONs with reduced anionic charge and potential for antisense applications.

Inclusion of methoxy groups inverts the thermodynamic stabilities of DNA–RNA hybrid duplexes: A molecular dynamics simulation study

Modified nucleic acids have found profound applications in nucleic acid based technologies such as antisense and antiviral therapies. Previous studies on chemically modified nucleic acids have suggested that modifications incorporated in furanose sugar especially at 2′-position attribute special properties to nucleic acids when compared to other modifications. 2′-O-methyl modification to deoxyribose sugars of DNA–RNA hybrids is one such modification that increases nucleic acid stability and has become an attractive class of compounds for potential antisense applications. It has been reported that modification of DNA strands with 2′-O-methyl group reverses the thermodynamic stability of DNA–RNA hybrid duplexes. Molecular dynamics simulations have been performed on two hybrid duplexes (DR and RD) which differ from each other and 2′-O-methyl modified counterparts to investigate the effect of 2′-O-methyl modification on their duplex stability. The results obtained suggest that the modification drives the conformations of both the hybrid duplexes towards A-RNA like conformation. The modified hybrid duplexes exhibit significantly contrasting dynamics and hydration patterns compared to respective parent duplexes. In line with the experimental results, the relative binding free energies suggest that the introduced modifications stabilize the less stable DR hybrid, but destabilize the more stable RD duplex. Binding free energy calculations suggest that the increased hydrophobicity is primarily responsible for the reversal of thermodynamic stability of hybrid duplexes. Free energy component analysis further provides insights into the stability of modified duplexes.

Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

Highly stable triple helix formation by homopyrimidine (L)-acyclic threoninol nucleic acids with single stranded DNA and RNA.

Acyclic (L)-threoninol nucleic acid (aTNA) containing thymine, cytosine and adenine nucleobases were synthesized and shown to form surprisingly stable triplexes with complementary single stranded homopurine DNA or RNA targets. The triplex structures consist of two (L)-aTNA strands and one DNA or RNA, and these triplexes are significantly stronger than the corresponding DNA or RNA duplexes as shown in competition experiments. As a unique property the (L)-aTNAs exclusively form triplex structures with DNA and RNA and no duplex structures are observed by gel electrophoresis. The results were compared to the known enantiomer (D)-aTNA, which forms much weaker triplexes depending upon temperature and time. It was demonstrated that (L)-aTNA triplexes are able to stop primer extension on a DNA template, showing the potential of (L)-aTNA for antisense applications.

Phosphorothioates, essential components of therapeutic oligonucleotides.

Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.

Comparison of duplex stabilizing properties of 2'-fluorinated nucleic acid analogues with furanose and non-furanose sugar rings.

We compare the duplex stabilizing properties of 2'-fluorinated nucleic acid analogues with furanose and non-furanose ring systems and dissect the relative contributions of hydration, sugar conformation, and fluorine configuration toward the overall T(m) value. We find that the stabilization imparted by fluorine substitution is additive over that obtained by restricting the conformation of the sugar ring itself. Our studies support further evaluation of fluorinated nucleic acid analogues with non-furanose sugar rings as surrogates of 2'-F RNA for therapeutic antisense applications.

Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications

A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications

Plasmonic laser treatment for Morpholino oligomer delivery in antisense applications

Several cell transfection techniques have been developed in the last decades for specific applications and for various types of molecules. In this context, laser based approaches are of great interest due to their minimal invasiveness and spatial selectivity. In particular, laser induced plasmon based delivery of exogenous molecules into cells can have great impact on future applications. This approach allows high-throughput laser transfection by excitation of plasmon resonances at gold nanoparticles non-specifically attached to the cell membrane. In this study, we demonstrate specific gene-knockdown by transfection of Morpholino oligos using this technique with optimized particle size. Furthermore, we evaluated the cytotoxicity of plasmonic laser treatment by various assays, including LDH activity and ROS formation. In summary, this study gives important insights into this new approach and clearly demonstrates its relevance for possible biological applications.

Antisense Oligonucleotides Therapy in the Treatment of Cerebral Gliomas: A Review

Patients affected by cerebral gliomas, despite classical strategies adopted, show a very poor prognosis. Current treatment consists of regimens that include surgical debulking, radiation therapy, and systemic chemotherapy. However, the median survival after surgery and radiation therapy alone is 9 months, and systemic chemotherapy is minimally effective. Advances in molecular biology have better depicted the mechanisms involved in the genesis of cerebral gliomas and identified specific gene sequences to be targeted in the malignant cell genome. Gene expression can be blocked using various strategies. The concept of antisense-mediated gene inhibition has now emerged as a potentially powerful alternative or adjunct to conventional cancer chemotherapy. This strategy is able to block selectively glioma cells which interfer to gliomagenesis molecular pathways. The antisense molecules, delivered inside the brain, penetrate into glioma cells blocking specific genic functions. Antisense oligonucleotides are complementary to the target mRNA and this bind cause the block and/or the reduction of the encoded protein synthesis. Genes coding for growth factors and their receptors, proto-oncogenes, cellular proteases, kinases, and proteins important in cell cycle control and apoptosis represent ideal target for antisense oligonucleotides treatment. In this study, we report the most relevant findings of antisense oligonucleotides application in gliomas treatment.

Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide Modifier

Anti-microRNA oligonucleotides (AMOs) are steric blocking antisense reagents that inhibit microRNA (miRNA) function by hybridizing and repressing the activity of a mature miRNA. First generation AMOs employed 2′-O-Methyl RNA nucleotides (2′OMe) with phosphorothioate (PS) internucleotide linkages positioned at both ends to block exonuclease attack. Second generation AMOs improved potency through the use of chemical modifications that increase binding affinity to the target, such as locked nucleic acid (LNA) residues. However, this strategy can reduce specificity as high binding affinity compounds can bind to and suppress function of related sequences even if one or more mismatches are present. Further, unnatural modified nucleic acid residues can have toxic side effects. In the present study, a variety of non-nucleotide modifiers were screened for utility in steric blocking antisense applications. A novel compound, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”), was discovered that increased binding affinity and blocked exonuclease degradation when placed at or near each end of a single-stranded oligonucleotide. This new modification was combined with the 2′OMe RNA backbone to make ZEN-AMOs. The new ZEN-AMOs have high potency and can effectively inhibit miRNA function in vitro at low nanomolar concentrations, show high specificity, and have low toxicity in cell culture.

Histone methylase MLL1 has critical roles in tumor growth and angiogenesis and its knockdown suppresses tumor growth in vivo

Mixed lineage leukemias (MLL) are human histone H3 lysine-4 specific methyl transferases that play critical roles in gene expression, epigenetics, and cancer. Herein, we demonstrated that antisense-mediated knockdown of MLL1 induced cell cycle arrest and apoptosis in cultured cells. Intriguingly, application of MLL1-antisense specifically knocked down MLL1 in vivo and suppressed the growth of xenografted cervical tumor implanted in nude mouse. MLL1-knockdown downregulated various growth and angiogenic factors such as HIF1α, VEGF and CD31 in tumor tissue affecting tumor growth. MLL1 is overexpressed along the line of vascular network and localized adjacent to endothelial cell layer expressing CD31, indicating potential roles of MLL1 in vasculogenesis. MLL1 is also overexpressed in the hypoxic regions along with HIF1α. Overall, our studies demonstrated that MLL1 is a key player in hypoxia signaling, vasculogenesis, and tumor growth, and its depletion suppresses tumor growth in vivo, indicating its potential in novel cancer therapy.

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7days to 8weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1-2days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.