G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.

Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

Composites of Nucleic Acids and Boron Clusters (C2B10H12) as Functional Nanoparticles for Downregulation of EGFR Oncogene in Cancer Cells.

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO's properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.

Preclinical development of a miR-132 inhibitor for heart failure treatment.

Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.

Boron clusters as a platform for new materials: composites of nucleic acids and oligofunctionalized carboranes (C2B10H12) and their assembly into functional nanoparticles.

Nucleic acids are key biomolecules in all life forms. These biomolecules can encode and transfer information via Watson-Crick base-pairing interactions and can form double-stranded structures between complementary sequences with high precision. These properties make nucleic acids extremely successful in applications in materials science as nanoconstruction materials. Herein, we describe a method for the automated synthesis of "oligopeds", which are building blocks based on the boron cluster structure equipped with short DNA adapters; these building blocks assemble into functional nanoparticles. The obtained, well defined, torus-like structures are the first DNA nanoconstructs based on a boron cluster scaffold. The results indicate the potential of boron clusters in DNA nanoconstruction and open the way for the design of entirely new types of buildings blocks based on polyhedral heteroborane geometry and its unique properties. The use of antisense oligonucleotides as DNA adapters illustrates one of the possible applications of the obtained nanoconstructs as vectors for therapeutic nucleic acids.

Antisense oligonucleotides targeting angiotensinogen: insights from animal studies.

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

Vitamin B12 transports modified RNA into E. coli and S. Typhimurium cells.

Specifically designed, antisense oligonucleotides are promising candidates for antibacterial drugs. They suppress the correct expression of bacterial genes by complementary binding to essential sequences of bacterial DNA or RNA. The main obstacle in fully utilizing their potential as therapeutic agents comes from the fact that bacteria do not uptake oligonucleotides from their environment. Herein, we report that vitamin B12 can transport oligonucleotides into Escherichia coli and Salmonella typhimurium cells. 5'-Aminocobalamin with an alkyne linker and azide-modified oligonucleotides enabled the synthesis of vitamin B12-2'OMeRNA conjugates using an efficient "click" methodology. Inhibition of protein expression in E. coli and S. Typhimurium cells indicates an unprecedented transport of 2'OMeRNA oligomers into bacterial cells via the vitamin B12 delivery pathway.

Modulation of blood-brain barrier function by a heteroduplex oligonucleotide in vivo.

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3'-hydroxylase from potato (Solanum tuberosum).

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato.

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Managing the sequence-specificity of antisense oligonucleotides in drug discovery.

All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.

Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes.

BACKGROUND/AIMS: Ginsenoside Rb1 (GS-Rb1) is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I) injuried cardiomyocytes. MATERIALS AND METHODS: Primary cultures of neonatal rat cardiomyocytes (NRCMs) was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. RESULTS: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK) mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. CONCLUSIONS: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in cardiomyocytes.

Qiliqiangxin Attenuates Phenylephrine-Induced Cardiac Hypertrophy through Downregulation of MiR-199a-5p.

BACKGROUND/AIMS: Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA. METHODS: The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and ß-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes. RESULTS: PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes. CONCLUSION: QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.

Impact of different cell penetrating peptides on the efficacy of antisense therapeutics for targeting intracellular pathogens.

There is a pressing need for novel and innovative therapeutic strategies to address infections caused by intracellular pathogens. Peptide nucleic acids (PNAs) present a novel method to target intracellular pathogens due to their unique mechanism of action and their ability to be conjugated to cell penetrating peptides (CPP) to overcome challenging delivery barriers. In this study, we targeted the RNA polymerase α subunit (rpoA) using a PNA that was covalently conjugated to five different CPPs. Changing the conjugated CPP resulted in a pronounced improvement in the antibacterial activity observed against Listeria monocytogenes in vitro, in cell culture, and in a Caenorhabditis elegans (C. elegans) infection model. Additionally, a time-kill assay revealed three conjugated CPPs rapidly kill Listeria within 20 minutes without disrupting the bacterial cell membrane. Moreover, rpoA gene silencing resulted in suppression of its message as well as reduced expression of other critical virulence genes (Listeriolysin O, and two phospholipases plcA and plcB) in a concentration-dependent manner. Furthermore, PNA-inhibition of bacterial protein synthesis was selective and did not adversely affect mitochondrial protein synthesis. This study provides a foundation for improving and developing PNAs conjugated to CPPs to better target intracellular pathogens.

MicroRNA-181 targets Yin Yang 1 expression and inhibits cervical cancer progression.

Dysregulated expression of microRNAs (miRNAs) has been observed in numerous types of human cancer, including cervical cancer (CC). The present study aimed to elucidate the expression and roles of miR­181 in cervical cancer tissues and cells. HeLa cells with a stable overexpression of miR­181 were generated and injected subcutaneously into the front legs of nude mice. Functional assays revealed a reduced rate of proliferation and an enhanced rate of apoptosis following transfection of CC cells with miR­181 mimics. In addition, miR­181 also suppressed tumor growth in the nude mice. At the molecular level, it was found that Yin Yang 1, an oncogene in several types of human cancer, was negatively regulated by miR­181. Therefore, the findings of the present study suggest that exogenous overexpression of miR­181 may be a potential approach for the treatment of CC in the future.

Both neuropeptide Y knockdown and Y1 receptor inhibition modulate CART-mediated appetite control.

Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

The interactions of amphiphilic antisense oligonucleotides with serum proteins and their effects on in vitro silencing activity.

Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.