Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules is one of the most promising approaches for delivering siRNA in vivo. The rate of molecular weight-dependent siRNA renal clearance is critical for the efficiency of this process. In this study, we prepared cholesterol-containing supramolecular complexes containing from three to eight antisense strands and examined their accumulation and silencing activity in vitro and in vivo. We have shown for the first time that such complexes with 2'F, 2'OMe, and LNA modifications exhibit interfering activity both in carrier-mediated and carrier-free modes. Silencing data from a xenograft tumor model show that 4 days after intravenous injection of cholesterol-containing monomers and supramolecular trimers, the levels of MDR1 mRNA in the tumor decreased by 85% and 68%, respectively. The in vivo accumulation data demonstrated that the formation of supramolecular structures with three or four antisense strands enhanced their accumulation in the liver. After addition of two PS modifications at the ends of antisense strands, 47% and 67% reductions of Ttr mRNA levels in the liver tissue were detected 7 days after administration of monomers and supramolecular trimers, respectively. Thus, we have obtained a new type of RNAi inducer that is convenient for synthesis and provides opportunities for modifications.

siRNA-Mediated Timp1 Silencing Inhibited the Inflammatory Phenotype during Acute Lung Injury.

Acute lung injury is a complex cascade process that develops in response to various damaging factors, which can lead to acute respiratory distress syndrome. Within this study, based on bioinformatics reanalysis of available full-transcriptome data of acute lung injury induced in mice and humans by various factors, we selected a set of genes that could serve as good targets for suppressing inflammation in the lung tissue, evaluated their expression in the cells of different origins during LPS-induced inflammation, and chose the tissue inhibitor of metalloproteinase Timp1 as a promising target for suppressing inflammation. We designed an effective chemically modified anti-TIMP1 siRNA and showed that Timp1 silencing correlates with a decrease in the pro-inflammatory cytokine IL6 secretion in cultured macrophage cells and reduces the severity of LPS-induced acute lung injury in a mouse model.

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. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

Folate-Equipped Cationic Liposomes Deliver Anti-MDR1-siRNA to the Tumor and Increase the Efficiency of Chemotherapy.

In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O'-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action.

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. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.

Immunostimulating RNA Delivered by P1500 PEGylated Cationic Liposomes Limits Influenza Infection in C57Bl/6 Mice.

The emergence of highly pathogenic viruses and a high speed of infection spread put forward the problem of the development of novel antivirals and their delivery vehicles. In this study, we investigated the antiviral effect of the previously identified immunostimulatory 19-bp dsRNA (isRNA) with 3'-nucleotide overhangs, which stimulates interferon α synthesis when delivered using cationic liposomes consisting of 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride and lipid-helper dioleoylphosphatidylethanolamine and its PEGylated formulation P1500 in vitro and in vivo. In vitro data showed that isRNA/2X3-DOPE complexes protected L929 cells from encephalomyocarditis virus infection, while isRNA/P1500 complexes were not active, which correlates with their lower transfection activity in cell culture. Comparison of the interferon-inducing activity of isRNA in BALB/c, CBA and C57Bl/6 mice showed that PEGylated liposomes significantly enhance the interferon-inducing activity of isRNA in vivo. The antiviral efficacy of the isRNA in vivo was considerably affected by the delivery system. The cationic liposomes 2X3-DOPE did not enhance the antiviral properties of isRNA in vivo. Similar liposomes equipped with a PEGylated lipoconjugate provided a pronounced anti-influenza effect of the isRNA in vivo. Administration of isRNA to C57Bl/6 led to a decrease in virus titers in the lungs and a significant decrease in the severity of the infection. Administration of a similar formulation to BALB/c mice caused only a mild antiviral effect at the initial stages of the infection. The data show that isRNA in combination with the PEGylated delivery system can be considered an effective means of suppressing influenza A infection.

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. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.

Trimeric Small Interfering RNAs and Their Cholesterol-Containing Conjugates Exhibit Improved Accumulation in Tumors, but Dramatically Reduced Silencing Activity.

Cholesterol derivatives of nuclease-resistant, anti-MDR1 small-interfering RNAs were designed to contain a 2'-OMe-modified 21-bp siRNA and a 63-bp TsiRNA in order to investigate their accumulation and silencing activity in vitro and in vivo. The results showed that increasing the length of the RNA duplex in such a conjugate increases its biological activity when delivered using a transfection agent. However, the efficiency of accumulation in human drug-resistant KB-8-5 cells during delivery in vitro in a carrier-free mode was reduced as well as efficiency of target gene silencing. TsiRNAs demonstrated a similar biodistribution in KB-8-5 xenograft tumor-bearing SCID mice with more efficient accumulation in organs and tumors than cholesterol-conjugated canonical siRNAs; however, this accumulation did not provide a silencing effect. The lack of correlation between the accumulation in the organ and the silencing activity of cholesterol conjugates of siRNAs of different lengths can be attributed to the fact that trimeric Ch-TsiRNA lags mainly in the intercellular space and does not penetrate sufficiently into the cytoplasm of the cell. Increased accumulation in the organs and in the tumor, by itself, shows that using siRNA with increased molecular weight is an effective approach to control biodistribution and delivery to the target organ.

Are Small Nucleolar RNAs "CRISPRable"? A Report on Box C/D Small Nucleolar RNA Editing in Human Cells.

CRISPR technologies are nowadays widely used for targeted knockout of numerous protein-coding genes and for the study of various processes and metabolic pathways in human cells. Most attention in the genome editing field is now focused on the cleavage of protein-coding genes or genes encoding long non-coding RNAs (lncRNAs), while the studies on targeted knockout of intron-encoded regulatory RNAs are sparse. Small nucleolar RNAs (snoRNAs) present a class of non-coding RNAs encoded within the introns of various host genes and involved in post-transcriptional maturation of ribosomal RNAs (rRNAs) in eukaryotic cells. Box C/D snoRNAs direct 2'-O-methylation of rRNA nucleotides. These short RNAs have specific elements in their structure, namely, boxes C and D, and a target-recognizing region. Here, we present the study devoted to CRISPR/Cas9-mediated editing of box C/D snoRNA genes in Gas5. We obtained monoclonal cell lines carrying mutations in snoRNA genes and analyzed the levels of the mutant box C/D snoRNA as well as the 2'-O-methylation status of the target rRNA nucleotide in the obtained cells. Mutations in SNORD75 in the obtained monoclonal cell line were shown to result in aberrant splicing of Gas5 with exclusion of exons 3 to 5, which was confirmed by RT-PCR and RNA-Seq. The obtained results suggest that SNORD75 contains an element for binding of some factors regulating maturation of Gas5 pre-lncRNA. We suggest that METTL3/METTL14 is among such factors, and m6A-methylation pathways are involved in regulation of Gas5 splicing. Our results shell light on the role of SNORDs in regulating splicing of the host gene.

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)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

Current Development of siRNA Bioconjugates: From Research to the Clinic.

Small interfering RNAs (siRNAs) acting via RNA interference mechanisms are able to recognize a homologous mRNA sequence in the cell and induce its degradation. The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degradation of siRNAs by nucleases in biological fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo (e.g., viruses, cationic lipids, polymers, nanoparticles), but all have limitations for therapeutic use. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i.e., the covalent connection of siRNAs with biogenic molecules (lipophilic molecules, antibodies, aptamers, ligands, peptides, or polymers). Bioconjugates are "ideal nanoparticles" since they do not need a positive charge to form complexes, are less toxic, and are less effectively recognized by components of the immune system because of their small size. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use.

Extra Purified Exosomes from Human Placenta Contain An Unpredictable Small Number of Different Major Proteins.

Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

Molecular Mechanism of the Antiproliferative Activity of Short Immunostimulating dsRNA.

Small double-stranded RNAs with certain sequence motifs are able to interact with pattern-recognition receptors and activate the innate immune system. Recently, we identified a set of short double-stranded 19-bp RNA molecules with 3-nucleotide 3'-overhangs that exhibited pronounced antiproliferative activity against cancer cells in vitro, and antitumor and antimetastatic activities in mouse models in vivo. The main objectives of this study were to identify the pattern recognition receptors that mediate the antiproliferative action of immunostimulating RNA (isRNA). Two cell lines, epidermoid carcinoma KB-3-1 cells and lung cancer A549 cells, were used in the study. These lines respond to the action of isRNA by a decrease in the growth rate, and in the case of A549 cells, also by a secretion of IL-6. Two sets of cell lines with selectively silenced genes encoding potential sensors and signal transducers of isRNA action were obtained on the basis of KB-3-1 and A549 cells. It was found that the selective silencing of PKR and RIG-I genes blocked the antiproliferative effect of isRNA, both in KB-3-1 and A549 cells, whereas the expression of MDA5 and IRF3 was not required for the antiproliferative action of isRNA. It was shown that, along with PKR and RIG-I genes, the expression of IRF3 also plays a role in isRNA mediated IL-6 synthesis in A549 cells. Thus, PKR and RIG-I sensors play a major role in the anti-proliferative signaling triggered by isRNA.

Fluorophore Labeling Affects the Cellular Accumulation and Gene Silencing Activity of Cholesterol-Modified siRNAs In Vitro.

The objective of this study was to analyze the effects of fluorophores on the intracellular accumulation and biological activity of small interfering RNA (siRNA) and its cholesterol conjugates. In this study, we used stem-loop real-time PCR and calibration curves to quantitate cellular siRNA accumulation. Attachment of fluorophores significantly affected both the accumulation and biological activity of siRNA conjugates. The severity of this effect depended significantly on the structure of the conjugate; fluorophores (Cy5.5 or Alexa-488) attached to siRNA, facing the side of the duplex opposite to cholesterol, enhanced the unproductive intracellular accumulation of the conjugate when delivered in carrier-free mode. Enhanced cellular accumulation of siRNA conjugates did not result in enhanced biological activity of the conjugate. Moreover, the attachment of a hydrophobic fluorophore, such as Cy5.5, to conventional siRNA also enhanced its apparent intracellular accumulation, but not its biological activity. Thus, the use of fluorescent labels for the study of the intracellular accumulation of siRNA and its conjugates formed with different molecules is possible only for a limited range of structures, and requires verification using alternative methods.

Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins.

Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018.

Applications of Bacteriophages in the Treatment of Localized Infections in Humans.

In the recent years, multidrug-resistant bacteria have become a global threat, and phage therapy may to be used as an alternative to antibiotics or, at least, as a supplementary approach to treatment of some bacterial infections. Here, we describe the results of bacteriophage application in clinical practice for the treatment of localized infections in wounds, burns, and trophic ulcers, including diabetic foot ulcers. This mini-review includes data from various studies available in English, as well as serial case reports published in Russian scientific literature (with, at least, abstracts accessible in English). Since, it would be impossible to describe all historical Russian publications; we focused on publications included clear data on dosage and rout of phage administration.

Profiling of 179 miRNA Expression in Blood Plasma of Lung Cancer Patients and Cancer-Free Individuals.

Lung cancer is one of major cancers, and survival of lung cancer patients is dictated by the timely detection and diagnosis. Cell-free circulating miRNAs were proposed as candidate biomarkers for lung cancer. These RNAs are frequently deregulated in lung cancer and can persist in bodily fluids for extended periods of time, shielded from degradation by membrane vesicles and biopolymer complexes. To date, several groups reported the presence of lung tumour-specific subsets of miRNAs in blood. Here we describe the profiling of blood plasma miRNAs in lung cancer patients, healthy individuals and endobronchitis patients using miRCURY LNA miRNA qPCR Serum/Plasma Panel (Exiqon). From 241 ratios differently expressed between cancer patients and healthy individuals 19 miRNAs were selected for verification using the same platform. LASSO-penalized logistic regression model, including 10 miRNA ratios comprised of 14 individual miRNAs discriminated lung cancer patients from both control groups with AUC of 0.979.

Nuclease-resistant 63-bp trimeric siRNAs simultaneously silence three different genes in tumor cells.

We designed a multimeric nuclease-resistant 63-bp trimeric small-interfering RNA (tsiRNA) comprising in one duplex the sequence of siRNAs targeting mRNAs of MDR1, LMP2, and LMP7 genes. We show that such tsiRNA is able to suppress the expression of all the target genes independently and with high efficiency, acting via a Dicer-dependent mechanism. tsiRNA is diced into 42- and 21-bp duplexes inside the cell. tsiRNA-induced gene silencing is characterized by kinetics similar to that of canonical siRNA, while the silencing efficiency is significantly higher than that of canonical siRNA with the same sequence.

Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene.

Chemical modifications are an effective way to improve the therapeutic properties of small interfering RNAs (siRNAs), making them more resistant to degradation in serum and ensuring their delivery to target cells and tissues. Here, we studied the carrier-free biodistribution and biological activity of a nuclease-resistant anti-MDR1 cholesterol-siRNA conjugate in healthy and tumor-bearing severe combined immune deficiency (SCID) mice. The attachment of cholesterol to siRNA provided its efficient accumulation in the liver and in tumors, and reduced its retention in the kidneys after intravenous and intraperitoneal injection. The major part of cholesterol-siRNA after intramuscular and subcutaneous injections remained in the injection place. Confocal microscopy data demonstrated that cholesterol-siRNA spread deep in the tissue and was present in the cytoplasm of almost all the liver and tumor cells. The reduction of P-glycoprotein level in human KB-8-5 xenograft overexpressing the MDR1 gene by 60% was observed at days 5-6 after injection. Then, its initial level recovered by the eighth day. The data showed that, regardless of the mode of administration (intravenous, intraperitoneal, or peritumoral), cholesterol-siMDR efficiently reduced the P-glycoprotein level in tumors. The designed anti-MDR1 conjugate has potential as an adjuvant therapeutic for the reversal of multiple drug resistance of cancer cells.