Mariya I. Meschaninova

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Small Interfering RNA Targeted to IGF-IR Delays Tumor Growth and Induces Proinflammatory Cytokines in a Mouse Breast Cancer Model

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2′-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2′-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

Modeling of Antigenomic Therapy of Mitochondrial Diseases by Mitochondrially Addressed RNA Targeting a Pathogenic Point Mutation in Mitochondrial DNA

Background: Point mutations in mitochondrial genome cause severe clinical disorders. Results: We designed recombinant RNA molecules imported into mitochondria of human cells, which are able to decrease the proportion of mitochondrial DNA molecules bearing a pathogenic point mutation. Conclusion: Imported recombinant RNAs can function as anti-replicative agents in human mitochondria. Significance: This is a new approach for therapy of mitochondrial diseases. Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene.

Silencing activity of 2′-O-methyl modified anti-MDR1 siRNAs with mismatches in the central part of the duplexes

The thermodynamic properties of siRNA duplexes are important for their silencing activity. siRNAs with high thermodynamic stability of both the central part of the duplex and in the whole, usually display low silencing activity. Destabilization of the central part of the siRNA duplex could increase its silencing activity. However, mismatches located in the central part of the duplex could substantially decrease the amount of RNAi efficacy, hindering active RISC formation and function. In this study, we examined the impact of duplex destabilization by nucleotide substitutions in the central part (7–10 nt counting from the 5′‐end of the antisense strand) of the nuclease‐resistant siRNA on its silencing activity.

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.

Multipyrene tandem probes for point mutations detection in DNA.

Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems.

[Cholesterol-modified anti-MDR1 small interfering RNA: uptake and biological activity].

Small interfering RNAs (siRNA) are considered to be potential agents for specific gene silencing, but low the efficacy of siRNA delivery into cells limits their biomedical application. Accumulation of siRNA coupled with cholesterol residue at the 5′-end of the “sense” strand (chol-siRNA) was studied in HEK293, HepG2, SC1, and KB-8-5 cells. In the absence of a transfection agent, the levels of both carrier-free and chol-siRNAs were very low, whereas transfection agent substantially increased transfection rate in all cell lines; in HEK293, SC1, and KB-8-5 cells transfection efficiency of the chol-siRNA was higher than that of the corresponding unmodified siRNA. Biological activity of anti-MDR1-siRNAs targeted to the 557–577 nt region of the MDR1 gene mRNA was estimated as multiple drug resistance phenotype reverting activity of KB-8-5 cancer cells. The chol-siRNA induced cancer cells’ death in the presence of previously tolerated vinblastine doses more effectively than unmodified siRNA.

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.

Impact of chemical modifications in the structure of isRNA on its antiproliferative and immunostimulatory properties

Short double-stranded RNAs, depending on their structure, sequence, and the method of delivery to cells, can activate the system of innate and adaptive immunity. The immunostimulatory activity of nucleic acids can be used in antitumor and antiviral therapy. We have previously identified a biologically active immunostimulatory 19-bp dsRNA (isRNA) with 3′-nucleotide overhangs, which inhibits the proliferation of tumor cells, stimulates interferon synthesis, and suppresses the growth and metastasis of tumors. Here, we have studied the impact of chemical modifications in the isRNA structure and the type of the transfection agent on the antiproliferative and immunostimulatory properties of isRNA. It has been shown that the attachment of an aminohexyl group or a cholesterol residue to the 5′-terminus of the first strand of the isRNA duplex does not impair its antiproliferative and immunostimulatory properties in vitro and in vivo when it is used in complex with a transfection agent, whereas the modification at the 5′-end of the second strand has an adverse effect on the biological activity of isRNA. It has been found that, when used without the transfection agent, isRNA conjugated with a cholesterol residue does not display antiproliferative and immunostimulatory properties. The use of isRNA in complex with low-toxicity liposomes 2Х3-DOPE enhances its activity: the intravenous injection of the isRNA/2Х3-DOPE complex induces a 55-fold increase in the level of interferon α (INF-α) in the murine blood 6 h after the injection, which is significantly higher than the INF-α level after the injection of the isRNA/Lipofectamine 2000 complex. The cytoplasmic localization of isRNA is crucially important for the manifestation of its antiproliferative activity since the selective inhibition of the dsRNA cytoplasmic sensor PKR (dsRNA-dependent protein kinase R) by 2-aminopurine completely blocks the antiproliferative effect of isRNA.