Tatyana O. Kabilova

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.

Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice.

Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3’- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma.

Targeted delivery of nucleic acids into xenograft tumors mediated by novel folate-equipped liposomes

Graphical abstract Figure. No Caption available. Abstract Folate receptors (FR) are cellular markers highly expressed in various cancer cells. Here, we report on the synthesis of a novel folate‐containing lipoconjugate (FC) built of 1,2‐di‐O‐ditetradecyl‐rac‐glycerol and folic acid connected via a PEG spacer, and the evaluation of the FC as a targeting component of liposomal formulations for nucleic acid (NA) delivery into FR expressing tumor cells. FR‐targeting liposomes, based on polycationic lipid 1,26‐bis(cholest‐5‐en‐3&bgr;‐yloxycarbonylamino)‐7,11,16,20‐tetraazahexacosan tetrahydrochloride (2X3), lipid helper dioleoylphosphatidylethanolamine (DOPE) and novel FC, formed small compact particles in solution with diameters of 60 ± 22 nm, and were not toxic to cells. Complexes of NAs with the liposomes were prepared at various nitrogen to phosphate ratios (N/P) to optimize liposome/cell interactions. We showed that FR‐mediated delivery of different nucleic acids mediated by 2X3‐DOPE/FC liposomes occurs in vitro at low N/P (1/1 and 2/1); under these conditions FC‐containing liposomes display 3–4‐fold higher transfection efficiency in comparison with conventional formulation. Lipoplexes formed at N/P 1/1 by targeted liposomes and cargo (Cy7‐labeled siRNA targeting MDR1 mRNA) in vivo efficiently accumulate in tumor (˜15–18% of total amount), and kidneys (71%), and were retained there for more than 24 h, causing efficient downregulation of p‐glycoprotein expression (to 40% of control) in tumors. Thus, FC containing liposomes provide effective targeted delivery of nucleic acids into tumor cells in vitro and in xenograft tumors in vivo.

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.

Polycationic amphiphiles based on triethylenetetramine and their transfection efficacy

Novel polycationic amphiphiles derived from triethylenetetramine, a spermine analogue, containing cholesterol or dialkylglycerol residues as hydrophilic domains were synthesized. The amphiphiles and a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) served for the preparation of cationic liposomes, physicochemical properties of which were evaluated. A comparative study of cytotoxicity and transfection efficiency demonstrated that the replacement of spermine by triethylenetetramine decreased transfection activity of cationic liposomes.