Extracellular vesicle mimetics as delivery vehicles for oligonucleotide-based therapeutics and plasmid DNA.

Introduction: Small membrane particles called extracellular vesicles (EVs) transport biologically active cargo between cells, providing intercellular communication. The clinical application of EVs is limited due to the lack of scalable and cost-effective approaches for their production and purification, as well as effective loading strategies.

Methods: Here we used EV mimetics produced by cell treatment with the actin-destabilizing agent cytochalasin B as an alternative to EVs for the delivery of therapeutic nucleic acids.

Engineering supramolecular dynamics of self-assembly and turnover of oncogenic microRNAs to drive their synergistic destruction in tumor models.

Rationally-engineered functional biomaterials offer the opportunity to interface with complex biology in a predictive, precise, yet dynamic way to reprogram their behaviour and correct shortcomings. Success here may lead to a desired therapeutic effect against life-threatening diseases, such as cancer. Here, we engineered "Crab"-like artificial ribonucleases through coupling of peptide and nucleic acid building blocks, capable of operating alongside and synergistically with intracellular enzymes (RNase H and AGO2) for potent destruction of oncogenic microRNAs.

Single Shot vs. Cocktail: A Comparison of Mono- and Combinative Application of miRNA-Targeted Mesyl Oligonucleotides for Efficient Antitumor Therapy.

Rational combinations of sequence-specific inhibitors of pro-oncogenic miRNAs can efficiently interfere with specific tumor survival pathways, offering great promise for targeted therapy of oncological diseases. Herein, we uncovered the potential of multicomponent therapy by double or triple combinations of highly potent mesyl phosphoramidate (µ) antisense oligodeoxynucleotides targeted to three proven pro-oncogenic microRNAs-miR-17, miR-21, and miR-155. A strong synergism in the inhibition of proliferation and migration of B16 melanoma cells was demonstrated in vitro for pairs of µ-oligonucleotides, which resulted in vivo in profound inhibition (up to 85%) of lung metastases development after intravenous injection of µ-oligonucleotide-transfected B16 cells in mice.

Bulge-Forming miRNases Cleave Oncogenic miRNAs at the Central Loop Region in a Sequence-Specific Manner.

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)Gly] (BC-miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC-miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA.

Heteroleptic Pd(II) and Pt(II) Complexes with Redox-Active Ligands: Synthesis, Structure, and Multimodal Anticancer Mechanism.

A series of heteroleptic square-planar Pt and Pd complexes with bis(diisopropylphenyl) iminoacenaphtene (dpp-Bian) and Cl, 1,3-dithia-2-thione-4,5-dithiolate (dmit), or 1,3-dithia-2-thione-4,5-diselenolate (dsit) ligands have been prepared and characterized by spectroscopic techniques, elemental analysis, X-ray diffraction analysis, and cyclic voltammetry (CV). The intermolecular noncovalent interactions in the crystal structures were assessed by density functional theory (DFT) calculations. The anticancer activity of Pd complexes in breast cancer cell lines was limited by their solubility.

Antisense oligonucleotide gapmers containing phosphoryl guanidine groups reverse MDR1-mediated multiple drug resistance of tumor cells.

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences.

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g.

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency.

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins.

Transport Oligonucleotides-A Novel System for Intracellular Delivery of Antisense Therapeutics.

Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized.

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance.

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs.

Catalytic Knockdown of miR-21 by Artificial Ribonuclease: Biological Performance in Tumor Model.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)Gly] capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides.

Enhanced Inhibition of Tumorigenesis Using Combinations of miRNA-Targeted Therapeutics.

The search for effective strategies to inhibit tumorigenesis remains one of the most relevant scientific challenges. Among the most promising approaches is the direct modulation of the function of short non-coding RNAs, particularly miRNAs. These molecules are propitious targets for anticancer therapy, since they perform key regulatory roles in a variety of signaling cascades related to cell proliferation, apoptosis, migration, and invasion.

Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties.

Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21.

Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H.

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3'-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH-Gly(ArgLeu)-TCAA displaying ribonuclease activity to its 5'-end.

The systemic tumor response to RNase A treatment affects the expression of genes involved in maintaining cell malignancy.

Recently, pancreatic RNase A was shown to inhibit tumor and metastasis growth that accompanied by global alteration of miRNA profiles in the blood and tumor tissue (Mironova et al., 2013). Here, we performed a whole transcriptome analysis of murine Lewis lung carcinoma (LLC) after treatment of tumor-bearing mice with RNase A.

[Inhibition of Invasive Properties of Murine Melanoma by Bovine Pancreatic DNase I In Vitro and In Vivo].

After a long pause, the accumulation of data on the involvement of tumor-specific DNA and extracellular DNA in metastasis has again placed enzymes with deoxyribonuclease activity in the focus of the search for antitumor and antimetastatic drugs. In this work, the ability of bovine pancreatic DNase I to reduce the invasive potential of B16 melanoma has been investigated in vitro and in vivo. It was found that DNase I had a cytotoxic effect on B16 melanoma cells (IC50 ≈ 10^(4) U/mL).

Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment.

Taking into account recently obtained data indicating the participation of circulating extracellular DNA (exDNA) in tumorigenesis, enzymes with deoxyribonucleic activity have again been considered as potential antitumour and antimetastatic drugs. Previously, using murine Lewis lung carcinoma and hepatocellular carcinoma A1 tumour models, we have shown the antimetastatic activity of bovine DNase I, which correlates with an increase of DNase activity and a decrease of exDNA concentration in the blood serum of tumour-bearing mice. In this work, using next-generation sequencing on the ABS SOLiD™ 5.

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells.

MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.

Ribonuclease binase decreases destructive changes of the liver and restores its regeneration potential in mouse lung carcinoma model.

The successful application of exogenous ribonucleases of different origin to suppress tumor growth allows one to consider them as perspective therapeutics for treatment of oncological diseases. An important aspect of the success of an anti-cancer drug is low hepatotoxicity, which will reduce, if not eliminate entirely the undesirable side effects of treatment. Previously we have shown that ribonuclease from Bacillus intermedius (binase) exhibits high antitumor and antimetastatic activity in tumor models of different histological origin.

MicroRNA drop in the bloodstream and microRNA boost in the tumour caused by treatment with ribonuclease A leads to an attenuation of tumour malignancy.

Novel data showing an important role of microRNAs in mediating tumour progression opened a new field of possible molecular targets for cytotoxic ribonucleases. Recently, antitumour and antimetastatic activities of pancreatic ribonuclease A were demonstrated and here genome-wide profiles of microRNAs in the tumour and blood of mice bearing Lewis lung carcinoma after treatment with RNase A were analysed by high-throughput Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) sequencing technology. Sequencing data showed that RNase A therapy resulted in the boost of 116 microRNAs in tumour tissue and a significant drop of 137 microRNAs in the bloodstream that were confirmed by qPCR.

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells.

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS 40 and melanoma B-16.

The Toxic Effects of Polychemotherapy onto the Liver Are Accelerated by the Upregulated MDR of Lymphosarcoma.

Antitumor therapy of hematological malignancies is impeded due to the high toxicity of polychemotherapy toward liver and increasing multiple drug resistance (MDR) of tumor cells under the pressure of polychemotherapy. These two problems can augment each other and significantly reduce the efficiency of antineoplastic therapy. We studied the combined effect of polychemotherapy and upregulated MDR of lymphosarcoma RLS(40) onto the liver of experimental mice using two treatment schemes.

Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein.

RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofluorometry and electrophoresis. Experiments on RLS(40) tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specific small interfering RNA (siRNA).

Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I.

Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.