Elena L. Chernolovskaya

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.

Silencing of MDR 1 gene in cancer cells by siRNA

Inhibition of p‐glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB‐8‐5 cells by synthetic 21‐bp double‐stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.

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.

Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference

In the present study, we have applied the siRNA approach to reduce the expression of AML1-ETO and RUNX1(K83N) oncogenes, which are frequently found in leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5′-untranslated region of mRNA for the mutant RUNX1(K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of oncogenes following the introduction of shRNAs into cells.

Immunoproteasome enhances intracellular proteolysis of myelin basic protein

300 Proteasome is a multisubunit protein complex that exhibits proteolytic activity and is present in the nuclei and cytoplasm of cells. The 20S proteasome, which has a molecular weight of 700 kDa and a sedimenta tion coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome [1]. The degradation of proteins in the cell is regulated by the ubiquitinylation system, which marks the old or defective protein molecules for their recognition by the proteasome and subsequent proteolysis [2]. One of the key biological functions of the proteasome is the hydrolysis of intracellular proteins to the antigenic peptides, which are then presented on the cell surface on the major histocompatibility complex class I mole cules [3]. Recent studies indicate the existence of a molecular mechanism by which the peptides gener ated by the proteasome can also be presented on the major histocompatibility complex class II molecules [4]. The catalytic activity of a constitutive proteasome is mediated by three subunits, β1, β2, and β5, which are constitutively expressed in cells. The proteasome, which contains corresponding immunosubunits β1i, β2i, and β5i the catalytic center, is called the immuno proteasome and is significantly different from the con stitutive proteasome in its activity and substrate speci ficity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome [5, 6]. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic pro teins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell [7]. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies [8], rheu matoid arthritis [9], autoimmune colitis [10], Alzhe imer’s disease [11], and Huntington disease [12]). One of the most common and socially significant autoimmune diseases is multiple sclerosis (MS), which is characterized by the destruction of the myelin sheath of nerve fibers. Myelin basic protein (MBP) is a major autoantigen in multiple sclerosis. At present, the molecular mechanisms underlying the develop ment of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immuno proteasome in the development of this disease [13]. Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomy elitis (EAE), an experimental model of MS [14]. Dur ing further development of this research, we created a model to study the intracellular proteolysis of MBP in mammalian cells. Here we show that the intracellular hydrolysis of MBP is significantly accelerated when the proteasome–immunoproteasome balance is shifted toward the latter. It is known that the expression of the myelin basic protein in mammals is detected solely in the central and peripheral nervous systems. This protein is local ized in the membrane of specialized cells, oligoden drocytes and Schwann cells, forming the myelin sheath of axons. Unfortunately, work with primary human neuronal cultures is associated with numerous experimental difficulties (first of all, the lack of mate rial and ethical concerns). In view of this, at the first step of this work, to study the proteolysis of MBP ex vivo we created a genetic construct that made it possible to express a recombinant human MBP in mammalian cells. For this purpose, a DNA fragment 546 bp long, encoding the full length MBP, was amplificated by PCR using specific overlapping oligo nucleotides and then cloned into the pBudCE4.1/EF Immunoproteasome Enhances Intracellular Proteolysis of Myelin Basic Protein

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment.MethodsThe transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA.ResultsIn the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3′-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC.ConclusionsThe obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Arrest of cancer cell proliferation by dsRNAs

Abstract:  Inhibition of c‐myc and N‐myc genes by dsRNAs in carcinoma and neuroblastoma cells was investigated. siRNA‐Ex3 targeted to the third exon of c‐myc gene was found to decrease the level of c‐myc but not N‐myc mRNA and decrease the rate or even arrest proliferation of c‐myc overexpressing cell lines KB‐3‐1 and SK‐N‐MC. This siRNA did not affect proliferation of IMR‐32 (which overexpress N‐myc). siRNA‐Ex2 corresponding (with 1‐2 mismatches) to the conservative region of the second exon of both c‐ and N‐myc was able to downregulate both genes and to reduce proliferation of KB‐3‐1, SK‐N‐MC, and IMR‐32 cells. Long dsRNA corresponding to the 3 exon of c‐myc gene (dsMyc), poly(I:C), and GU‐rich siRNA‐I, corresponding to the intron sequence of human MDR1 gene demonstrated high antiproliferative activity in experiments with KB‐3‐1 cells. Short‐term elevation of PKR or/and OAS1 mRNA levels was detected in the cells affected by interferon inducer poly(I:C). dsMyc, poly(I:C), and even siRNA‐I, which could not affect c‐myc mRNA by RNA interference mechanism, were found to inhibit proliferation of the KB‐3‐1 cells and to decrease the mRNA level of interferon‐sensitive genes c‐myc and β‐actin.

Inhibition of human cancer-cell proliferation by long double-stranded RNAs.

Three different enzymatically synthesized long double-stranded RNAs (dsRNAs) [448 bp homologous to the third exon of c-myc messenger RNA (mRNA) (dsMyc); 473 bp homologous to enhanced green fluorescent protein (EGFP) mRNA (dsEGFP) and control interferon inducer poly(I:C)] were studied for antiproliferative and gene-silencing activities in KB-3-1, SK-N-MC, and IMR-32 human cancer cell lines. Simple incubation with these dsRNAs did not affect the expression of c-myc gene and the proliferation of KB-3-1 and IMR-32 cells, but inhibited the proliferation of SK-N-MC cells. Transfection of KB-3-1 and SK-N-MC cells using Oligofectamine-dsRNAs complexes resulted in dose-dependent inhibition of c-myc and beta-actin genes expression and proliferation. The data show that dsMyc, acting both as interferon inducer and as gene-specific interfering RNA, is more effective as c-myc inhibitor than other tested dsRNAs. The most efficient inhibition of proliferation was displayed by dsEGFP RNA, dsMyc and poly(I:C) were effective only when used in higher concentrations. Our data indicate that transfection of studied dsRNAs causes an increase in apoptotic and dead cells number in the cell population. This proapoptotic activity correlates with dsRNAs-induced antiproliferative activity. However the difference in cell growth between dsRNA-treated and Oligofectamine-only treated cells can not be attributed only to the loss of cells due to the apoptosis; it also indicates some retardation of cell cycle progression caused by dsRNA.

Silencing of c‐myc Expression in Tumor Cells by siRNA

Suppression of c‐myc protooncogene expression in KB‐3‐1 cells by siRNA was investigated. The siRNA duplex targeted to the exon 3 of c‐myc mRNA was prepared by in vitro transcription with T7 RNA polymerase on short dsDNA‐templates. It was found that incubation of KB‐3‐1 cells in the presence of 75 nM siRNA results in decrease of the c‐myc mRNA level down to 5% of the level in the control cells and significant decline of KB‐3‐1 cell proliferation rate. Using 200 nM siRNA four‐fold decrease of KB‐3‐1 cells proliferation rate was observed and this effect was stable at least 96 h after transfection.

42‐ and 63‐bp anti‐MDR1‐siRNAs bearing 2′‐OMe modifications in nuclease‐sensitive sites induce specific and potent gene silencing

DsRNAs longer than 30 bp induce interferon response and global changes in gene expression profile in mammalians. 21 bp siRNA and 25/27 bp dsiRNA acting via RNA interference mechanism are used for specific gene silencing in this class of organisms. We designed selectively 2′‐O‐methyl‐modified 42 and 63 bp anti‐MDR1‐siRNAs that silence the expression of P‐glycoprotein and restore the sensitivity of drug‐resistant cancer cells to cytostatic more efficiently than canonical 21 bp siRNAs. We also show that they act in a Dicer‐independent mode and are devoid of immunostimulating properties. Our findings suggest that 42 and 63 bp siRNAs could be used as potential therapeutics.