Antisense Technology Research Articles

Lipoprotein (a), coronary artery disease risk, and MACE in individuals without standard modifiable risk factors in the UK Biobank

Abstract Background A significant proportion of patients with coronary artery disease (CAD) lack standard modifiable risk factors (SMuRFs; smoking, hypercholesterolaemia, diabetes and hypertension), for example with recent analyses demonstrating that around 25% of patients with ST-elevation myocardial infarction are SMuRF-less(1). With continued focus on primary prevention in cardiovascular disease, the SMuRF-less subset is likely to increase over time. A body of evidence has implicated lipoprotein (a) in CAD, but no such studies have investigated this association in a purely SMuRF-less population. Purpose To investigate the role of lipoprotein (a) in predicting CAD risk and major adverse cardiovascular events (MACE) in a large, SMuRF-less cohort. Methods The UK Biobank is a large prospective multicentre study which recruited over 500,000 participants in the UK aged 37-73 years at entry between 2006-2010. SMuRFs were defined as 1) smoking (current or historical), 2) hypercholesterolaemia – lipid lowering medication on enrolment or total cholesterol >5.5mmol/L or LDL-C >3.5mmol/L, 3) previous diagnosis of diabetes mellitus, or HbA1c >48mmol/mol, 4) previous diagnosis of hypertension. MACE was defined as a composite of coronary artery disease – previous myocardial infarction, percutaneous coronary intervention or coronary artery bypass graft surgery –, stroke, or all-cause mortality. Results Of the total UK Biobank cohort, 46,139 participants were identified as SMuRF-less together with a valid lipoprotein (a) level. Median follow-up duration was 12yrs. In univariate analyses (Table 1), there was no significant difference in lipoprotein (a) concentrations between individuals with and without MACE (16.9 vs. 17.1nmol/L respectively, p = 0.913). The overall event rate was low, with just 3% MACE (1381 events) overall. Older age, male gender, apolipoprotein A, apolipoprotein B, HbA1c level, HDL cholesterol level and body mass index were all associated with MACE. In regard to all-cause mortality alone, there was no association with lipoprotein (a) level, although the other aforementioned parameters were all significantly associated with this outcome. Similarly, lipoprotein (a) was not a significant predictor of stroke. However, there was a large difference in lipoprotein (a) levels between patients with and without CAD (27.2 vs. 17.1nmol/L respectively, p = 0.004). In Kaplan-Meier analysis, lipoprotein (a) level above the 77th percentile (60.3nmol/L) was significantly associated with CAD (p=0.032). In Cox regression models, despite the low event rate, lipoprotein (a) was a significant predictor of CAD (HR 1.00-1.01, p=0.01), and importantly the only potentially modifiable parameter (N = 39,433, 111 events, Table 2). Conclusion Lipoprotein (a) independently predicts CAD in those without SMuRFs, highlighting the importance of checking this parameter in such patients, particularly given the novel antisense technologies in development which may lower lipoprotein (a)(2).Univariate analysesCox regression for CAD

Chemical strategies for antisense antibiotics.

Antibacterial resistance is a severe threat to modern medicine and human health. To stay ahead of constantly-evolving bacteria we need to expand our arsenal of effective antibiotics. As such, antisense therapy is an attractive approach. The programmability allows to in principle target any RNA sequence within bacteria, enabling tremendous selectivity. In this Tutorial Review we provide guidelines for devising effective antibacterial antisense agents and offer a concise perspective for future research. We will review the chemical architectures of antibacterial antisense agents with a special focus on the delivery and target selection for successful antisense design. This Tutorial Review will strive to serve as an essential guide for antibacterial antisense technology development.

Synergistic pH-responsive MUC-1 aptamer-conjugated Ag/MSN Janus nanoparticles for targeted chemotherapy, photothermal therapy, and gene therapy in breast cancer

Drug resistance in cancer treatment, primarily attributed to the overexpression of the multidrug resistance (MDR) gene, significantly hampers the effectiveness of chemotherapy. This mechanism, driven by the increased production of P-glycoprotein (P-gp) efflux pumps, highlights the urgent need for innovative strategies to combat drug resistance in cancer patients. This study explores the application of antisense technology to suppress MDR gene expression, while addressing the challenges of instability and limited cellular uptake associated with antisense oligonucleotides. We synthesized Janus silver-mesoporous silica nanoparticles (Ag/MSN JNPs) using a sol-gel method, characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), revealing uniformly sized, dumbbell-shaped nanoparticles with an average size of 285 ± 5.12 nm. Doxorubicin (DOX) was loaded into the porous structure of the mesoporous silica, and JNPs were functionalized with chitosan (CS) to incorporate P-gp antisense and a MUC-1 aptamer, serving as a pH-responsive gatekeeper. Our findings indicate that the Ap-As-DOX-JNPs achieved a remarkable 89 ± 0.59 % cell death in drug-resistant MCF-7/ADR cells after 48 h, alongside an 80 % reduction in P-gp expression. The combination of DOX, antisense technology, and photothermal therapy utilizing these JNPs demonstrates a promising strategy to effectively overcome drug resistance. Notably, normal MCF-7 cells exhibited reduced viability from 39.11 ± 1.12 % to 30.05 ± 1.07 % when treated with DOX-JNPs under near-infrared (NIR) irradiation. These results underscore the potential of utilizing MUC-1 aptamer-conjugated Janus nanoparticles in conjunction with chitosan as a gatekeeper to enhance the efficacy of chemotherapy, photothermal therapy, and gene therapy in overcoming multidrug resistance in cancer treatment.

8817 IGF-II Regulates Magmas A Mitochondrial Protein Required For Cell Viability And ROS Protection

Abstract Disclosure: K. Granados: None. A. Durán: None. F. Almaguel: None. D. De Leon: None. Our laboratory previously showed that Insulin-like growth factor II (IGF-II) promotes cell proliferation, inhibits apoptosis and induces chemoresistance of Breast Cancer (BC) cells. We demonstrated that IGF-II achieves this by inhibiting pro-apoptotic proteins and stimulating anti-apoptotic proteins. Those studies also demonstrated that IGF-II prevented mitochondrial induced apoptosis by inhibiting depolarization of the mitochondrial membrane. Thus, IGF-II regulates the mitochondria to prevent cell death and to promote tumor growth and chemoresistance. Since IGF-II regulates mitochondrial proteins, we decided to assess how IGF-II regulates MAGMAS, a protein located in the inner mitochondrial membrane that is a critical component of a complex that controls mitochondrial protein import and ROS. Of note, MAGMAS was recently identified as being highly expressed in breast cancer, prostate cancer and during early fetal development which parallels IGF-II expression. Immunostaining of TNBC showed high levels of IGF-II and MAGMAS. Thus, our hypothesis is that IGF-II regulates MAGMAS and IGF-II secreting breast cancer cells will express higher levels of MAGMAS. Our preliminary results of rtPCR and Western blotting experiments validated our hypothesis demonstrating that IGF-II regulated the transcription of MAGMAS. To characterize the mechanisms by which IGF-II regulates MAGMAS, we chose the triple negative breast cancer (TNBC) cell line CRL-2335 which we demonstrated produces high levels of IGF-II and MAGMAS. CRL2335 cells were established from an AA TNBC patient and they express high levels of IGF-II and MAGMAS. Confocal microscopy and Western blotting and cellular fractionation were used to assess IGF-II regulation of MAGMAS in TNBC cells. The expression of MAGMAS was analyzed in wild CRL-2335, and IGF-II antisense transfected CRL-2335 cells and compared to cells treated with IGF-II. Results showed that CRL-2335 express high levels of MAGMAS in the mitochondria and blocking the expression of IGF-II by antisense technology significantly decreased MAGMAS. Exogenous IGF-II treatment to the antisense cells increased MAGMAS as demonstrated by Immunofluorescence and WB. Since IGF-II increases the localization of MAGMAS to the mitochondria we propose that IGF-II regulates MAGMAS to protect cancer cells from ROS induced cell death and chemoresistance. Completion of this study will validate IGF-II as a regulator of MAGMAS. Validation of IGF-II regulation of MAGMAS represents an important new tool for the treatment of TNBC. Presentation: 6/2/2024

INTASYL self-delivering RNAi decreases TIGIT expression, enhancing NK cell cytotoxicity: a potential application to increase the efficacy of NK adoptive cell therapy against cancer

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance, they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors, which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), a well-known checkpoint receptor involved in T-cell exhaustion, has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here, we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability, are rapidly and efficiently taken up by cells, and can be easily incorporated into cell product manufacturing. INTASYL PH-804, which targets TIGIT, suppresses TIGIT mRNA and protein expression in NK cells, resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition, thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy, particularly for hematological malignancies.

Improving organoleptic and antioxidant properties by inhibition of novel miRstv_7 to target key genes of steviol glycosides biosynthetic pathway in Stevia rebaudiana Bertoni.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties.

Synthesis of N3-Methyluridine- and 2'-O-Alkyl/2'-Fluoro-N3-Methyluridine-Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides.

In this article, we describe the synthesis of N3-methyluridine (m3U) and 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2'-O-alkyl-m3U phosphoramidite starts with commercially available uridine to achieve a tritylated m3U intermediate, followed by 2'-O-alkylation and finally phosphitylation. Synthesis of the 2'-F-m3U phosphoramidite is obtained from a commercially available 2'-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2'-position of m3U that can be employed in numerous nucleic acid-based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2'-O-alkyl-N3-methyluridine analogs and their corresponding phosphoramidites Alternate Protocol 1: Synthesis of 2'-O-TBDMS-N3-methyluridine and its phosphoramidite Alternate Protocol 2: Synthesis of 2'-fluoro-N3-methyluridine and its phosphoramidite Basic Protocol 2: Solid-phase synthesis of N3-methyluridine-modified DNA and RNA oligonucleotides.

Targeting Oligodendrocyte Dynamics and Remyelination: Emerging Therapies and Personalized Approaches in Multiple Sclerosis Management.

Multiple sclerosis [MS] is a progressive autoimmune condition that primarily affects young people and is characterized by demyelination and neurodegeneration of the central nervous system [CNS]. This in-depth review explores the complex involvement of oligodendrocytes, the primary myelin- producing cells in the CNS, in the pathophysiology of MS. It discusses the biochemical processes and signalling pathways required for oligodendrocytes to function and remain alive, as well as how they might fail and cause demyelination to occur. We investigate developing therapeutic options that target remyelination, a fundamental component of MS treatment. Remyelination approaches promote the survival and differentiation of oligodendrocyte precursor cells [OPCs], restoring myelin sheaths. This improves nerve fibre function and may prevent MS from worsening. We examine crucial parameters influencing remyelination success, such as OPC density, ageing, and signalling pathway regulation [e.g., Retinoid X receptor, LINGO-1, Notch]. The review also examines existing neuroprotective and antiinflammatory medications being studied to see if they can assist oligodendrocytes in surviving and reducing the severity of MS symptoms. The review focuses on medicines that target the myelin metabolism in oligodendrocytes. Altering oligodendrocyte metabolism has been linked to reversing demyelination and improving MS patient outcomes through various mechanisms. We also explore potential breakthroughs, including innovative antisense technologies, deep brain stimulation, and the impact of gut health and exercise on MS development. The article discusses the possibility of personalized medicine in MS therapy, emphasizing the importance of specific medicines based on individual molecular profiles. The study emphasizes the need for reliable biomarkers and improved imaging tools for monitoring disease progression and therapy response. Finally, this review focuses on the importance of oligodendrocytes in MS and the potential for remyelination therapy. It also underlines the importance of continued research to develop more effective treatment regimens, taking into account the complexities of MS pathology and the different factors that influence disease progression and treatment.

Calcium-Enhanced Medium-Based Delivery of Splice Modulating Antisense Oligonucleotides in 2D and 3D hiPSC-Derived Neuronal Models.

Antisense technology demonstrates significant potential for addressing inherited brain diseases, with over a dozen products already available and numerous others in the development pipeline. The versatility of differentiating induced pluripotent stem cells (iPSCs) into nearly all neural cell types proves invaluable for comprehending the mechanisms behind neurological diseases, replicating cellular phenotypes, and advancing the testing and development of new therapies, including antisense oligonucleotide therapeutics. While delivering antisense oligonucleotides (ASOs) to human iPSC-based neuronal models has posed challenges, this study explores various delivery methods, including lipid-based transfection, gymnotic uptake, Ca(2+)-enhanced medium (CEM)-based delivery, and electroporation, in 2D and 3D hiPSC-derived neuronal models. This study reveals that CEM-based delivery exhibits efficiency and low toxicity in both 2D neuronal cultures and 3D brain organoids. Furthermore, the findings indicate that CEM is slightly more effective in neurons than in astrocytes, suggesting promising avenues for further exploration and optimization of preclinical ASO strategies in the treatment of neurological disorders.

Novel Therapeutic Horizons: SNCA Targeting in Parkinson's Disease.

Alpha-synuclein (αSyn) aggregates are the primary component of Lewy bodies, which are pathological hallmarks of Parkinson's disease (PD). The toxicity of αSyn seems to increase with its elevated expression during injury, suggesting that therapeutic approaches focused on reducing αSyn burden in neurons could be beneficial. Additionally, studies have shown higher levels of SNCA mRNA in the midbrain tissues and substantia nigra dopaminergic neurons of sporadic PD post-mortem brains compared to controls. Therefore, the regulation of SNCA expression and inhibition of αSyn synthesis could play an important role in the pathogenesis of injury, resulting in an effective treatment approach for PD. In this context, we summarized the most recent and innovative strategies proposed that exploit the targeting of SNCA to regulate translation and efficiently knock down cytoplasmatic levels of αSyn. Significant progress has been made in developing antisense technologies for treating PD in recent years, with a focus on antisense oligonucleotides and short-interfering RNAs, which achieve high specificity towards the desired target. To provide a more exhaustive picture of this research field, we also reported less common but highly innovative strategies, including small molecules, designed to specifically bind 5'-untranslated regions and, targeting secondary nucleic acid structures present in the SNCA gene, whose formation can be modulated, acting as a transcription and translation control. To fully describe the efficiency of the reported strategies, the effect of αSyn reduction on cellular viability and dopamine homeostasis was also considered.

Tailored antisense oligonucleotides designed to correct aberrant splicing reveal actionable groups of mutations for rare genetic disorders

Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2′-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.

Engineering erucic acid biosynthesis in camelina (Camelina sativa) via FAE1 gene cloning and antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistanceto abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme β-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.

Silencing the fatty acid elongase gene elovl6 induces reprogramming of nutrient metabolism in male Oreochromis niloticus

Elongation of very long-chain fatty acids protein 6 (ELOVL6) plays a pivotal role in the synthesis of endogenous fatty acids, influencing energy balance and metabolic diseases. The primary objective of this study was to discover the molecular attributes and regulatory roles of ELOVL6 in male Nile tilapia, Oreochromis niloticus. The full-length cDNA of elovl6 was cloned from male Nile tilapia, and was determined to be 2255-bp long, including a 5′-untranslated region of 193 bp, a 3′-untranslated region of 1252 bp, and an open reading frame of 810 bp encoding 269 amino acids. The putative protein had typical features of ELOVL proteins. The transcript levels of elovl6 differed among various tissues and among fish fed with different dietary lipid sources. Knockdown of elovl6 in Nile tilapia using antisense RNA technology resulted in significant alterations in hepatic morphology, long-chain fatty acid synthesis, and fatty acid oxidation, and led to increased fat deposition in the liver and disrupted glucose/lipid metabolism. A comparative transcriptomic analysis (elovl6 knockdown vs. the negative control) identified 5877 differentially expressed genes with significant involvement in key signaling pathways including the peroxisome proliferator-activated receptor signaling pathway, fatty acid degradation, glycolysis/gluconeogenesis, and the insulin signaling pathway, all of which are crucial for lipid and glucose metabolism. qRT-PCR analyses verified the transcript levels of 13 differentially expressed genes within these pathways. Our findings indicate that elovl6 knockdown in male tilapia impedes oleic acid synthesis, culminating in aberrant nutrient metabolism.

Advances in Breeding for Oil Quality Enhancement in Indian Mustard (Brassica spp. L.): Achievements, Challenges, and Research Opportunities

Major objectives in oil crop improvement are enhancement of seed and oil yield, quality of oil according to its use, i.e. edible or industrial uses, breeding of varieties that fit in different cropping systems and breeding biotic and abiotic stress resistant/tolerant varieties. Despite traditional breeding approaches, including pure line breeding, yielding only modest gains in productivity, recent advancements in mustard breeding have led to significant breakthroughs in both productivity and oil quality. This review discusses the innovative breeding strategies that have contributed to these advancements, with a focus on hybrid development, oil quality enhancement, and biotechnological approaches. To enhance productivity, researchers at the University of Delhi have developed hybrid seed production techniques using transgenic Barnase-barstar systems and cytoplasmic male sterility (CMS) systems. These systems enable large-scale hybrid seed production, with field trials demonstrating significant yield heterosis ranging from 31% to 55% compared to national check varieties. In addition to productivity, improving oil and meal quality has been a key objective. By integrating genes from canola-quality mustard lines, breeders have achieved reductions in erucic acid and glucosinolates, enhancing the health profile and industrial applicability of mustard oil. A high-density linkage map developed using an F1 double haploid mapping population has facilitated the marker-assisted backcross breeding of desirable traits, enabling precise transfer of key quality traits. Transgenic approaches, such as antisense RNA technology, have led to the development of high-oleic, low-linoleic mustard lines with improved fatty acid profiles. These advancements reflect a strategic combination of conventional and biotechnological methods, demonstrating a clear pathway for boosting mustard yields while enhancing oil quality. Molecular markers reported for genetic diversity assessment, mapping and tagging genes/QTLs for different qualitative and quantitative traits and their use in marker-assisted selection have been presented. This progress not only addresses current challenges but also sets the stage for future research aimed at further optimizing productivity, oil quality, and resistance to pests and diseases in mustard cultivation.

Silencing of Resistance Mechanism in Vancomycin-Resistance Enterococci Using Antisense RNA of vanA Gene

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

A short review on CRP synthesis inhibition in cardiovascular disease

C-reactive Protein (CRP) is synthesized in the liver. Synthesis is stimulated via the IL-1ß/IL6 pathway. CRP activates the complement system via C1q and macrophages via Fcγ receptors. Since elevated CRP plasma levels are associated with increased cardiovascular risk, CRP may play a causal role in cardiovascular disease. One approach to transfer these observations into standard medical care would be to generate hepatic CRP synthesis inhibitors and use them in controlled clinical trials. Despite huge pharmacological efforts, the search for CRP synthesis inhibitors proved to be difficult. First, the antisense oligonucleotide RNA technology, although a promising idea, has not yet led to results feasible for clinical practice. Secondly, high throughput screening assays in search for hepatic CRP inhibitors were limited by the fact that primary human hepatocytes do not adequately grow in vitro. Use of genetically engineered hepatoma cells led to the observation that cardiac glycosides are capable of inhibiting CRP synthesis. Because of patent law considerations, however, pharmaceutical companies had limited interest in further pursuing this possible path. Upstream inhibition of IL-1ß and IL-6 by antibodies has shown positive results in cardiovascular clinical trials, but because of side effects none of these antibodies has yet received FDA approval. In contrast, long-term colchicine treatment, though not being a CRP-specific approach, has recently been approved by the FDA. Taken together, there is no compelling evidence until today that hepatic CRP synthesis can specifically, effectively and safely be inhibited in vivo in human medicine. Currently, other avenues appear more promising. Here, we summarize contemporary approaches to inhibit CRP synthesis and potential goals for future clinical trials.

General and Specific Cytotoxicity of Chimeric Antisense Oligonucleotides in Bacterial Cells and Human Cell Lines.

In the last two decades, antisense oligonucleotide technology has emerged as a promising approach to tackling various healthcare issues and diseases, such as antimicrobial resistance, cancer, and neurodegenerative diseases. Despite the numerous improvements in the structure and modifications of the antisense oligonucleotides (ASOs), there are still specific problems with their clinical efficacy and preclinical cytotoxicity results. To better understand the effects of the ASOs in this paper, we conducted many MTT assays to assess the general and specific cytotoxicity of four new chimeric ASOs in bacterial cells and human cell lines. We demonstrate the absence of inhibitory activity in the human pathogenic bacteria Staphylococcus aureus by non-specific ASOs. The pVEC-ASO1 and pVEC-ASO2 are designed to have no specific targets in S. aureus. They have only partial hybridization to the guanylate kinase mRNA. The pVEC-ASO3 targets UBA2 mRNA, a hallmark cancer pathology in MYC-driven cancer, while pVEC-ASO4 has no complementary sequences. We discovered some cytotoxicity of the non-specific ASOs in healthy and cancer human cell lines. The results are compared with two other ASOs, targeting specific mRNA in cancer cells. All ASOs are delivered into the cell via the cell-penetrating oligopeptide pVEC, which is attached to them. We draw a good correlation between the thermodynamic stability of ASO/target RNA and the toxicity effect in human cell lines. The data obtained signify the importance of thorough bioinformatic analysis and high specificity in designing and developing novel ASOs for safer therapeutic agents in clinical practice.

Clinical Progress in Hepatic Targeting for Novel Prophylactic Therapies in Hereditary Angioedema

Hereditary angioedema (HAE) is typically caused by a deficiency of the protease inhibitor C1 inhibitor (C1INH). The absence of C1INH activity on plasma kallikrein and factor XIIa leads to overproduction of the vasoactive peptide bradykinin, with resulting angioedema. As the primary site of C1INH and prekallikrein production, the liver is recognized as an important therapeutic target in HAE, leading to the development of hepatic-focused treatment strategies such as GalNAc-conjugated antisense technology and gene modification. This report reviews currently available data on hepatic-focused interventions for HAE that have advanced into human trials. Donidalorsen is an investigational GalNAc3-conjugated antisense oligonucleotide that binds to prekallikrein mRNA in the liver and reduces the expression of prekallikrein. Phase 2 data with subcutaneous donidalorsen demonstrated a significant reduction in HAE attack rate compared with placebo. Phase 3 trials are underway. ADX-324 is a GalNAc3-conjugated short-interfering RNA being investigated in HAE. BMN 331 is an investigational AAV5-based gene therapy vector that expresses wild-type human C1INH and is targeted to hepatocytes. A single intravenous dose of BMN 331 is intended to replace the defective SERPING1 gene and enable patients to produce functional C1INH. A first-in-human phase 1/2 study is ongoing with BMN 331. NTLA-2002 is an investigational invivo clustered regularly interspaced short palindromic repeats/Cas9-based therapy designed to knock outthe prekallikrein-coding KLKB1 gene in hepatocytes; a phase 1/2 study is ongoing. Findings from these and other ongoing studies are highly anticipated with the expectation of expanding the array of treatment options in HAE.

Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA.

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.