RESUMEN
Connective tissue growth factor (CTGF) is a multifunctional matricellular protein, playing a role as a central mediator in tissue remodeling and fibrosis. A number of reports have shown the pivotal roles of CTGF in the progression of fibrosis, suggesting CTGF as a promising therapeutic target for the treatment of fibrotic disorders including hypertrophic scars and keloids. In this study, we present the development of an interfering RNA molecule that efficiently inhibits the expression of CTGF via RNA interference mechanism both in vitro and in vivo. Chemical modifications were introduced to the asymmetric interfering RNA (asiRNA) backbone structure. The resulting RNA molecule, termed cell-penetrating asiRNA (cp-asiRNA), entered into cells and triggered RNA interference-mediated gene silencing without delivery vehicles. The gene-silencing activity of cp-asiRNA targeting CTGF (cp-asiCTGF) was examined both in vitro and in vivo. Furthermore, the administration of cp-asiCTGF in the rat skin excision wound model efficiently reduced the induction of CTGF and collagens during the wound-healing process. These results suggest that the cp-asiCTGF molecule could be developed into antifibrotic therapeutics such as antiscar drugs.
Asunto(s)
Cicatriz Hipertrófica/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , Animales , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patología , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Inmunohistoquímica , Masculino , Microscopía Confocal , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
RNA interference (RNAi) triggering oligonucleotides in unconventional structural format can offer advantages over conventional small interfering RNA (siRNA), enhanced cellular delivery and improved target gene silencing. With this concept, we present a well-defined tripodal-interfering RNA (tiRNA) structure that can induce simultaneous silencing of multiple target genes with improved potency. The tiRNA structure, formed by the complementary association of three single-stranded RNA units, was optimized for improved gene silencing efficacy. When combined with cationic polymers such as linear polyethyleneimine (PEI), tiRNA assembled to form a stable nano-structured complex through electrostatic interactions and induced stronger RNAi response over conventional siRNA-PEI complex. In combination with a liver-targeting delivery system, tripodal nucleic acid structure demonstrated enhanced fluorescent accumulation in mouse liver compared to standard duplex nucleic acid format. Tripodal RNA structure complexed with galactose-modified PEI could generate effective RNAi-mediated gene silencing effect on experimental mice models. Our studies demonstrate that optimized tiRNA structural format with appropriate polymeric carriers have immense potential to become an RNAi-based platform suitable for multi-target gene silencing.
Asunto(s)
Silenciador del Gen , Hígado/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Citocinas/metabolismo , Sistemas de Liberación de Medicamentos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Nanopartículas , Tamaño de la Partícula , Polietileneimina/química , ARN Interferente Pequeño/metabolismo , Distribución TisularRESUMEN
RNA interference (RNAi) is a highly efficient endogenous gene silencing mechanism mediated by short double-stranded RNAs termed small interfering RNAs (siRNAs). The current standard siRNA structure, which is used by most researchers to trigger sequence-specific target gene silencing, consists of a double strand region of 19 bp with 2 nt 3'-overhangs at both ends. However, in addition to the desired target gene silencing, this conventional siRNA structure also exhibits several unintended effects that constitute obstacles to the use of siRNA in gene function studies and therapeutics development. Here, we provide protocols for designing and preparing an alternative structure for RNAi trigger, termed asymmetric shorter-duplex RNA (asiRNA). The asiRNA structure has a duplex region shorter than 19 bp and has an asymmetric 3'-overhang structure. Importantly, the asiRNA structure not only triggers efficient target gene silencing comparable to that of the 19 bp standard siRNA structure but also significantly reduces nonspecific effects triggered by 19 bp siRNAs such as sense-strand-mediated off-target silencing and the saturation of RNAi machinery. Procedures are described for verifying that asiRNA activates gene silencing through an Ago2-dependent pathway and for assessing the miRNA pathway competition potency and specific and nonspecific silencing abilities of asiRNAs. We propose that asiRNA, an improved RNAi trigger that can overcome the nonspecific effects evoked by standard siRNA structures, can be developed as a precise and effective tool for both functional genomics and therapeutic applications.