Application Of Antisense Oligonucleotides Research Articles

Synthesis and evaluation of antisense oligonucleotides prodrug with G-quadruplex assembly and lysosome escape capabilities for oncotherapy

The applications of antisense oligonucleotides (ASOs) in rare or common diseases treatment have garnered great attention in recent years. Nevertheless, challenges associated with stability and bioavailability still persist, hampering the efficiency of ASOs. This work presents an ASO prodrug with parallel G-quadruplex assembly and lysosome escape capabilities for oncotherapy. Our findings revealed that the end-assembled quadruplex structure effectively shielded the ASO from enzymatic degradation. Meanwhile, the conjugation of maleimide within the quadruplex enhanced cellular uptake, potentially offering an alternative cell entry mechanism that circumvents lysosome involvement. Notably, an optimized molecule, Mal2-G4-ASO, exhibited remarkable therapeutic effects both in vitro and in vivo. This work presents a promising avenue for enhancing the activity of nucleic acid drugs in oncotherapy and potentially other disease contexts.

The application of antisense oligonucleotides in disease treatment

Scientific research in recent years has made every endeavor to combat against various untreatable diseases in neurodegenerative or genetic diseases under the guidance of constantly updated underlying gene-associated pathological mechanisms. However, substantial therapeutic challenges still remain and many original causes of diseases are under great debate. Problems of drugs for neurological disorders involve high resistance to drug molecules by the blood-brain barrier (BBB) and only work efficiently in certain populations of patients, which is also a side effect of genetic malady. The introduction of Antisense oligonucleotides (ASOs) brings a new era for these diseases and meets the requirement. The US Food and Drug Administration (FDA) has proven ten oligonucleotide drugs until 2020. This article reviews some of their clinical trials with mechanisms, up-to-date attempts in inventing new ASO therapy, continuous safety and efficacy in preclinical and clinical trials of proven ASO, and great potential usage for RNA virus therapy.

Engineering Antisense Oligonucleotides as Antibacterial Agents That Target FMN Riboswitches and Inhibit the Growth of Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli.

In the past several decades, antibiotic drug resistance has emerged as a significant challenge in modern medicine due to the rise of many bacterial pathogenic strains resistant to all known antibiotics. At the same time, riboswitches have emerged as novel targets for antibacterial drug discovery. Here for the first time, we describe the design and applications of antisense oligonucleotides as antibacterial agents that target a riboswitch. The antisense oligonucleotides are covalently coupled with two different cell-penetrating peptides, penetrating Gram-positive and Gram-negative bacterial cells. We specifically target Flavin MonoNucleotide (FMN) riboswitches in Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli that control both synthesis and import of FMN precursors. We have established an average antibiotic dosage by antisense oligonucleotides that inhibit 80% of bacterial growth at 700 nM (4.5 μg/mL). Furthermore, the antisense oligonucleotides do not exhibit toxicity in human cell lines at this concentration. The results demonstrate that riboswitches are suitable targets in antisense technology for antibacterial drug development.

Abstract B39: Modulation of insulin receptor alternative splicing to develop cancer therapeutics

Abstract Insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. While IN-RB has high affinity for insulin and much lower affinity for IGF1 or IGF2 ligands, IN-RA binds both insulin and IGF2 with high affinity and as such it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks such as proliferation and angiogenesis. Our data show that there is a significantly increased expression of IN-RA levels in multiple cancer patient cohorts, including rhabdomyosarcoma (RMS) and osteosarcoma (OS). We also found this increased IN-RA expression in tumor cell lines derived from multiple different pediatric tumor types such as RMS, OS, and Ewing’s sarcoma as compared to the control samples. Additionally, our data show that cellular stress such as hypoxia increases the expression of the IN-RA isoform. Adaptation to hypoxic environments is a hallmark of the neoplastic phenotype, and pediatric sarcomas exhibit an intrinsically hypoxic physiology. However, how the INR alternatively spliced mRNAs are regulated is not well understood, nor is the role that these isoforms play in the initiation and progression and therapeutic resistance of human cancer. Our central hypothesis is that hypoxia alters the expression of splicing factors, leading to the generation of the IN-RA isoform, which contributes to cancer progression beyond the micrometastatic stage. We used an insulin receptor mini-gene system to identify a binding site for the RNA binding protein cugbp, critical for the hypoxia-induced splicing regulation of IN-R. We targeted the cugbp binding site using an antisense oligonucleotide (ASO) walk to identify an ASO that binds to the cugbp binding site in the intron 10 of INR gene and modulates the levels of endogenous IN-RA. To allow the possibility for therapeutic intervention, we utilized this ASO in OS cell lines. We found that the application of ASO shifts the IN-R splicing towards IN-RB and significantly reduces proliferation, migration, and angiogenesis in cancer cell lines. In order to delineate the downstream consequences of this splicing change, we subjected the control and ASO-treated cells to mass spectrometric analysis. Preliminary analysis of this data reveals that proliferation markers such as Ki67, PI3-kinase pathway components, protein clusters such as cell-cell adhesion, and cell division, among others, are significantly downregulated in cells treated with ASO where the splicing has been restored to the IN-RB isoform. Our data open a new paradigm of how alternative splicing regulates cell signaling to mediate cancer-causing changes, and our ASO compounds show promising insight into how we can reinstate the splicing pattern and impede tumorigenesis. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Peter Houghton, Frank Rigo, Dawn Chandler. Modulation of insulin receptor alternative splicing to develop cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B39.

Phosphate-Methylated Oligonucleotides Past, Present and Future

At the moment, we see a great interest for application of Anti Sense Oligonucleotides (ASOs) in order to regulate the expression of genes related to certain diseases. These nucleotides obtained a number of fascinating properties by means of chemical manipulation of natural DNA and RNA under conservation of Watson-Crick base-pairing. About 35 years ago for our research in this field, we selected synthetically (short) phosphate-methylated DNA and RNA. It was concluded that there is an exclusive selection in hybridization affinity with natural DNA and RNA. These (bio)chemical and physical-chemical properties are extensively published. ASOs have found their way in public health as is clearly shown in the treatment of (progressive) neurological diseases. We focus specifically on the past, present and future of the phosphate-methylated oligonucleotides, illustrated with different research studies in chemistry and biophysics. A new field of application of modified DNAs is based on interactive improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing phosphate-methylated DNA as probe.

The Usher Syndrome, a Human Ciliopathy

The human Usher syndrome (USH) is a complex, rare disease manifesting in its most common form of inherited deaf-blindness. Due to the heterogeneous manifestation of the clinical symptoms, three clinical types (USH1-3) are distinguished according to the severity of the disease pattern. For a correct diagnosis, in addition to the auditory tests in early newborn screening, ophthalmological examinations and molecular genetic analysis are important. Ten known USH genes encode proteins, which are from heterogeneous protein families, interact in functional protein networks. In the eye and in the ear, USH proteins are expressed primarily in the mechano-sensitive hair cells and the rod and cone photoreceptor cells, respectively. In the hair cells, the USH protein networks are essential for the correct differentiation of the hair bundles as well as for the function of the mechano-electrical transduction complex in the matured cell. In the photoreceptor cells, USH proteins are located in the ciliary region and participate in intracellular transport processes. In addition, a USH protein network is present in the so-called calyceal processes. The lack of calyceal processes and the absence of a prominent visual phenotype in the mouse disqualifies mice as models for studies on the ophthalmic component of USH. While hearing impairments can be compensated with hearing aids and cochlear implants, there is no practical therapy for USH in the eye. Currently, gene-based therapy concepts, such as gene addition, applications of antisense oligonucleotides and TRIDs ("translational readthrough inducing drugs") for the readthrough of nonsense mutations are preclinically evaluated. For USH1B/MYO7A the UshStat gene therapy clinical trial is ongoing.

Morpholino-Mediated Exon Inclusion for SMA.

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. AONs with phosphorodiamidate morpholino oligomer(PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).

Epigenetic factors in atherogenesis: microRNA

MicroRNAs (miRNAs) are small (~22 nucleotides in length) noncoding RNA sequences regulating gene expression at posttranscriptional level. MicroRNAs bind complementarily to certain mRNA and cause gene silencing. The involvement of miRNAs in the regulation of lipid metabolism, inflammatory response, cell cycle progression and proliferation, oxidative stress, platelet activation, endothelial and vascular smooth muscle cells (VSMC) function, angiogenesis and plaque formation and rapture indicates important roles in the initiation and progression of atherosclerosis. The key role of microRNAs in pathophysiology of cardiovascular diseases (CVDs), including atherosclerosis, was demonstrated in recent studies. Creating antisense oligonucleotides is a novel technique for selective changes in gene expression both in vitro and in vivo. In this review, we draw attention to the role of miRNAs in atherosclerosis progression, using miRNA as the potential biomarkers and targets in the CVDs, as well as possible application of antisense oligonucleotides.

Abstract A73: Antisense oligonucleotides targeting ENO2 have selective anti-proliferative effects in glioblastoma cell with 1p36 genomic loss

Abstract Essential cellular processes are often supported by redundant genes and pathways. Cancer cells frequently harbor deletions or mutations in essential genes and survive because of this functional redundancy. Identification of recurrent events that leave cancer cells selectively sensitive to inhibition of a particular pathway or gene can provides the opportunity to develop therapeutics with a high degree of selectivity for cancer cells over normal cells. In Glioblastoma Multiform (GBM), one of the most malignant brain cancers, roughly 3-5% of tumors have loss of the chromosome locus 1p36. Among the genes lost at this locus, is enolase1 (ENO1). Loss of ENO1 is tolerated by the cancer cells and is not required for cancer initiation or progression; likely because of functional redundancy with the closely related paralog, ENO2. However, it has been recently demonstrated that cancer cells with homozygous ENO1 loss are selectively sensitive to ENO2 inhibition. Here we investigate the potential of Antisense Oligonucleotides (ASO) targeted to ENO2 as a therapeutic approach to treat GBM with 1p36 loss of ENO1.We show that constrained ethyl (cET) modified ASOs targeting ENO2 produced dose dependent inhibition of ENO2 RNA and protein levels in GBM cells when delivered by free uptake. ASO-mediated ENO2 depletion led to the selective inhibition of ENO1-null cells and not the isogenic cells engineered to express ENO1. Moreover, cell cycle analysis is demonstrated that ENO2 depletion resulted in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle. These findings support the application of ASO to elicit synthetic lethality in GBM tumors with 1p36 loss. Citation Format: Maria Shahmoradgoli, Curt Mazur, Youngsoo Kim, Brett P Monia, A. Robert Macleod, Florian M Muller, Christopher E Hart. Antisense oligonucleotides targeting ENO2 have selective anti-proliferative effects in glioblastoma cell with 1p36 genomic loss. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A73.

SM30 protein function during sea urchin larval spicule formation

A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins.

Antisense oligonucleotide suppression of serum amyloid A reduces amyloid deposition in mice with AA amyloidosis

AA amyloid patients who experience disease progression and develop renal failure have not received sufficient benefit from agents that treat inflammation or infection. We have begun to explore the potential application of antisense oligonucleotides (ASOs) to specifically suppress SAA production and thereby reduce amyloid deposition. Proof-of-concept experiments conducted in mice initially examined ASO ability to reduce serum levels of SAA during an acute inflammatory response. Peak SAA levels in ASO-treated mice were reduced as much as 65% relative to levels in saline-treated mice. The extent of suppression was dose-dependent and influenced by the time interval between ASO administration and inflammatory stimulation. Subsequent experiments tested whether ASO suppression of SAA was sufficient to mitigate amyloid deposition. Amyloidosis was induced by amyloid-enhancing factor and silver nitrate injection; ASO treatment was initiated 1 week later and continued 1× or 3× per week; inflammation was re-triggered by subsequent injection(s) of silver nitrate; mice were sacrificed after 4–5 weeks. Examination of tissues by Congo red staining and SAA/AA immunohistochemistry revealed consistently less amyloid in the organs of ASO-treated mice compared to saline-treated counterparts. These findings provide rationale for further investigation of SAA-specific ASOs as a potential therapy for AA amyloidosis.

Opportunities and challenges for the development of antisense treatment in neuromuscular disorders

Introduction: Neuromuscular disorders are diseases of the musculature and/or the nervous system, generally leading to loss of muscle function. They are a frequent cause of disability and treatment options are often only symptomatic. Interestingly, for a number of neuromuscular disorders the application of antisense oligonucleotides has therapeutic potential.Areas covered: The authors describe how this approach is exploited for different neuromuscular diseases, focusing on literature published in the past 10 years. For each disease the opportunities of this approach, the state of the art, and current challenges are described.Expert opinion: A lot of progress has been made in the development of antisense-mediated approaches during recent years and they may become clinically applicable in the near future.

Making sense of therapeutics using antisense technology

Introduction: Antisense oligonucleotides (ASOs) are short synthetic single-stranded DNA sequences that bind to and induce the cleavage of homologous stretches of mRNA sequences. These result in targeted destruction of mRNA and correction of genetic aberrations. ASOs thus can act as drug molecules and potentially rectify many disease conditions. The broad range of applications reported in the literature highlights the advances in the field.Areas covered: This review covers different areas in which use of ASOs has been shown to have therapeutic effects. Some drugs in different stages of preclinical and clinical trials are discussed in detail. The problems faced and the strategies to surmount them are also described. The readers will gain an understanding of the recent developments in the field of ASOs with emphasis on their therapeutic applications. They will also become aware of the different strategies used for targeted delivery of ASOs and their stabilization, which may be useful for their work in this field, or in the area of nucleic acid therapeutics in general.Expert opinion: The design and application of ASOs for recognition of target mRNA sequences have become a fairly straightforward protocol. The main problem lies in designing ASOs which are stable in in vivo milieu. The delivery and bioavailability of the oligonucleotide to the site of action continue to be hurdles in the development of ASOs and therapeutic molecules.

Antisense-Mediated RNA Targeting: Versatile and Expedient Genetic Manipulation in the Brain

A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

Therapeutic Applications of Antisense Oligonucleotides in Asthma and Allergy

Antisense oligonucleotides represent a novel therapeutic approach for manipulation of gene expression in the treatment of respiratory diseases. This methodology is based on the use of synthetic oligonucleotides to inhibit the expression of a specific target gene by inhibiting the function of the corresponding messenger RNA. Inhaled antisense oligonucleotides are therapeutic alternatives in the treatment of respiratory diseases including allergic asthma; however development of this technology is at an early stage and critical questions related to design, biological activity, and target delivery still remain. The present review will discuss the current status of antisense technology and its application in asthma and allergy. It will be focused in some representative patents including considerations on the future clinical prospects.

The Application of Antisense Oligonucleotides in the Gene Therapy of Duchenne Muscular Dystrophy

杜氏肌营养不良(DMD)是一种致死性X染色体连锁隐性遗传病,发病率高,患者多在30岁左右因心力衰竭和呼吸衰竭而死亡.近年来,在众多对DMD基因治疗方案中,反义寡核苷酸凭借其在肿瘤治疗和病毒控制中显示出的良好应用前景而受到研究人员的关注,他们利用反义寡核苷酸诱导基因中的特异外显子缺失以调控突变基因的表达,以此达到恢复具有功能的抗肌萎缩蛋白表达,并使DMD得到控制和治愈的目的。

Peripheral nerve injury decreased the expression of syntaxin1A mRNA and increased the frequency of mEPSCs in the mouse spinal dorsal horn

Peripheral nerve injury results in sprouting of nerve fibers in the spinal cord dorsal horn, which may result in neuropathic pain. It has been reported that rat dorsal root ganglion neurons showed an enhancement of axonal sprouting when expression of syntaxin1A, a SNARE protein that was shown to be involved in transmitter release, was inhibited by an application of antisense oligonucleotide. In the present study, we investigated the effects of peripheral nerve injury on the expression of syntaxin1A mRNA in the spinal cord dorsal horn using real time quantitative RT-PCR. We also recorded the miniature excitatory postsynaptic currents (mEPSCs) from neurons in the superficial dorsal horn neurons by patch clamp methods, and analyzed the effects of nerve injury on mEPSCs. Mechanical allodynia appeared immediately after nerve ligation and continued for 20 days, which was accompanied with the reduction of syntaxin1A mRNA expression. The frequency of mEPSCs significantly increased in the peripheral nerve-ligated mouse. This increase in the frequency of mEPSCs may underlie the cellular mechanism of allodynia development. The reduced expression of syntaxin1A concurrent with the allodynia and increased frequency of mEPSC may indicate a potential role for syntaxin1A in the development of allodynia, possibly through axonal sprouting in the spinal cord dorsal horn.

Correction of alternative splicing of tau in frontotemporal dementia and parkinsonism linked to chromosome 17.

Mutations in the human tau gene cause frontotemporal dementia and Parkinsonism associated with chromosome 17 (FTDP-17). One of the major disease mechanisms in FTDP-17 is the increased inclusion of tau exon 10 during pre-mRNA splicing. Here we show that modified oligonucleotides directed against the tau exon 10 splice junctions suppress inclusion of tau exon 10. The effect is mediated by the formation of a stable pre-mRNA-oligonucleotide hybrid, which blocks access of the splicing machinery to the pre-mRNA. Correction of tau splicing occurs in a tau minigene system and in endogenous tau RNA in neuronal pheochromocytoma cells and is specific to exon 10 of the tau gene. Antisense oligonucleotide-mediated exclusion of exon 10 has a physiological effect by increasing the ratio of protein lacking the microtubule-binding domain encoded by exon 10. As a consequence, the microtubule cytoskeleton becomes destabilized and cell morphology is altered. Our results demonstrate that alternative splicing defects of tau as found in FTDP-17 patients can be corrected by application of antisense oligonucleotides. These findings provide a tool to study specific tau isoforms in vivo and might lead to a novel therapeutic strategy for FTDP-17.

The concept and application of antisense oligonucleotides.

Since the identification of the double-stranded DNA helix by Watson and Crick in 1953, the knowledge of nucleotide structure and function has been an important potential tool in the study and therapy of disease. There is recent clinical evidence that antisense oligonucleotides may be important therapeutic compounds in the clinical therapy of a range of diseases, including infection (viruses and bacteria), oncology, and inflammation. Our laboratory-based understanding of antisense oligonucleotide activity has provided a foundation for their use in several human diseases. Potentially relevant applications include inflammatory bowel disease therapy, psoriasis, transplantation, rheumatoid arthritis, cytomegalovirus retinitis, hepatitis C, and solid tumor therapy. Here we will outline these applications as well as our ongoing clinical trials for Crohn's disease.

The role of CRH in behavioral responses to stress

Corticotropin-releasing hormone (CRH) and urocortin in the central nervous system affect behavior and can enhance behavioral responses to stressors. The action of CRH-related peptides is mediated through multiple receptors that differ markedly in their pharmacological profiles and anatomical distribution. Comparative pharmacology of CRH receptor agonists suggests that CRH, urocortin, sauvagine and urotensin consistently mimic, and CRH receptor antagonists consistently lessen, functional consequences of stressor exposure. Recently, important advances have been made in understanding the CRH system and its role in behavioral responses to stress by the development of specific CRH receptor antagonists, application of antisense oligonucleotides and development of transgenic mice lacking peptides and functional receptors. This review summarizes recent findings with respect to components of the CRH system and their role in stress-induced behavioral responses.