RESUMO
Double-stranded RNAs (dsRNAs) longer than 30 bp have not been considered desirable RNA interference (RNAi) triggering structures in mammalian cells as they nonspecifically activate innate immune response. However, in earlier studies, not only dsRNA length but also 5'-triphosphate moiety produced by in vitro transcription might have affected the stimulation of innate immune system. Herein, using chemically synthesized long dsRNAs without 5'-triphosphate, we elucidated direct relationship between length of dsRNAs and innate immune stimulation. First, we found that blunt-ended, chemically synthesized 38/40-60 bp-long dsRNAs induced retinoic acid-inducible gene I (RIG-I)-mediated innate immune response, which was suppressed by the introduction of the 2-nt 3' overhang structure. Surprisingly, we discovered that RIG-I activation by these long dsRNAs is also sequence dependent, and the sequence composition at dsRNA termini is important for RIG-I activation. In addition, we identified that long dsRNAs over 38 bp could elicit single- or dual-target gene silencing in a Dicer-independent manner. Taken together, our findings may serve as guidelines to develop an immunostimulatory RNAi trigger to exploit host's innate immune system, as well as a specific dual-gene targeting RNAi therapeutics platform without nonspecific innate immune stimulation by RIG-I.
Assuntos
Imunidade Inata , RNA de Cadeia Dupla , Animais , Proteína DEAD-box 58/genética , Inativação Gênica , Imunidade Inata/genética , Mamíferos/genética , Interferência de RNA , RNA de Cadeia Dupla/genéticaRESUMO
The efficient delivery of small interfering RNAs (siRNAs) to the target cells is critical for the pharmaceutical success of RNA interference (RNAi) drugs. One of the possible strategies to improve siRNA delivery is to identify auxiliary molecules that augment their cellular uptake. Herein, we performed a chemical library screening in an effort to discover small molecules that enhance the potency of cholesterol-conjugated, cell-penetrating asymmetric siRNAs (cp-asiRNAs). Interestingly, three compounds identified from the screen share a common dihydropyridine (DHP) core and function as L-type calcium channel blockers (CCBs). Using confocal microscopy and quantitative analysis of small RNAs, we demonstrated that the L-type CCBs increased the endocytic cellular uptake of cp-asiRNAs. Furthermore, these small molecules substantially improved the potency of cp-asiRNAs, not only in vitro but also in vivo on rat skin. Collectively, our study provides an alternative pharmacological approach for the identification of small molecules that potentiate the effects of therapeutic siRNAs.
Assuntos
Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/farmacocinética , Canais de Cálcio Tipo L/metabolismo , Di-Hidropiridinas/farmacocinética , Interferência de RNA , RNA Interferente Pequeno/farmacocinética , Animais , Biópsia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colesterol/química , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Di-Hidropiridinas/administração & dosagem , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/farmacocinética , Células HeLa , Humanos , Injeções Intradérmicas , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Pele/metabolismo , Pele/patologia , Bibliotecas de Moléculas Pequenas , TransfecçãoRESUMO
RNA interference (RNAi) offers great promise in life science research and therapeutic development, as it easily achieves a potent target gene knockdown with high specificity. Since the conventional small interfering RNA (siRNA) structure, known as 19 bp double-stranded RNA (dsRNA) with 2-nucleotide (nt) 3' overhang, has been introduced to successfully elicit the RNAi in mammalian cells, a variety of structural variants of RNAi trigger have been developed. Our group previously reported branched, tripodal interfering RNA (tiRNA) structures as a multigene targeting RNA structure inducing RNAi. However, the immune stimulatory effect of branched tiRNA structure has not been thoroughly evaluated. In this study, we show that tiRNA with blunt ends triggers innate immune response in T98G cell and mouse macrophage cells, which is dependent upon the retinoic acid-inducible gene I (RIG-I), a well-known cytoplasmic dsRNA sensor. Interestingly, immune response triggered by tiRNA can be suppressed by the introduction of 2-nt 3' overhang structure. Our finding expands the structural diversity of RIG-I ligands and provides a guide to develop a safe multitargeting RNA structure for therapeutic application.
Assuntos
RNA Helicases DEAD-box/genética , Imunidade Inata/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Animais , RNA Helicases DEAD-box/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Inativação Gênica , Células HeLa , Humanos , Camundongos , RNA de Cadeia Dupla/farmacologiaRESUMO
Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.
Assuntos
Proteínas Argonautas/genética , RNA Helicases DEAD-box/genética , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Linhagem Celular , RNA Helicases DEAD-box/deficiência , Genes Reporter , Células HeLa , Humanos , Luciferases/antagonistas & inibidores , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/deficiência , Survivina/antagonistas & inibidores , Survivina/genética , Survivina/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMO
AIM: Study was aimed at combining the advantages of nonclassical RNAi-triggering oligonucleotides with nanoparticle-based advanced delivery platforms for developing efficient therapeutic systems. MATERIALS & METHODS: We utilized a core-shell hybrid nanostructured platform for effectively delivering nonclassical RNAi triggers, namely long double stranded interfering RNA and tripodal interfering RNA. Core-shell structure was prepared by stably anchoring thiol-modified cationic polymer on the surface of growing crystal gold (Au) seeds, and the resulting particles were further complexed with nonclassical RNAi candidates via electrostatic interactions. RESULTS: Our studies clearly demonstrated that the unique combination of nonclassical RNAi structures with an advanced core-shell hybrid nanostructured platform is an effective module for advanced RNAi-based therapeutic development.
Assuntos
Iminas/química , Nanopartículas/química , Polietilenos/química , Polímeros/química , Terapêutica com RNAi/métodos , Animais , Transporte Biológico , Sobrevivência Celular , Inativação Gênica , Técnicas de Transferência de Genes , Ouro/química , Células HeLa , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/química , Imagem Óptica , Tamanho da Partícula , Interferência de RNA , Eletricidade Estática , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
Combination therapy involving the synergism between different therapeutic approaches seems to be a promising strategy in anticancer treatment. Immunostimulatory long interfering RNA (liRNA) structures capable of executing specific RNA interference (RNAi) mediated gene silencing tasks seem to be potential candidates for a combination approach. Apart from their therapeutic efficacy, the unique structural format of liRNA candidates facilitates better association with cationic polymers and significantly improves their intra-cellular delivery. In this study, we have developed a biocompatible cationic delivery platform based on low molecular weight branched polyethyleneimine-grafted-polyethylene glycol (bPEI-g-PEG) for advanced liRNA based anticancer therapy. With simple guanidine (GU) modification, the bPEI-g-PEG platform could induce a strong RNAi mediated response in cancer cells, without induction of any obvious toxicity. Moreover, liRNA complexed with GU-bPEI-g-PEG which targets expression of Survivin gene sensitized cancer cells for effective chemotherapy. A combination strategy involving immunostimulatory RNAi mediators with conventional chemotherapeutic drugs seems to be an effective approach in advanced anticancer treatment.
RESUMO
RNA oligonucleotides capable of inducing controlled immunostimulation combined with specific oncogene silencing via an RNA interference (RNAi) mechanism provide synergistic inhibition of cancer cell growth. With this concept, we previously designed a potent immunostimulatory long double stranded RNA, referred to as liRNA, capable of executing RNAi mediated specific target gene silencing. In this study, we developed a highly effective liRNA based targeted delivery system to apply in the treatment of glioblastoma multiforme. A stable nanocomplex was fabricated by complexing multimerized liRNA structures with cross-linked branched poly(ethylene imine) (bPEI) via electrostatic interactions. We show clear evidence that the cross-linked bPEI was quite effective in enhancing the cellular uptake of liRNA on U87MG cells. Moreover, the liRNA-PEI nanocomplex provided strong RNAi mediated target gene silencing compared to that of the conventional siRNA-PEI complex. Further, the bPEI modification strategy with specific ligand attachment assisted the uptake of the liRNA-PEI complex on the mouse brain endothelial cell line (b.End3). Such delivery systems combining the beneficial elements of targeted delivery, controlled immunostimulation, and RNAi mediated target silencing have immense potential in anticancer therapy.