Selection and Optimization of Asymmetric siRNA Targeting the Human c-MET Gene

Jo, Seul-gi; Hong, Sun Woo; Yoo, Jae Wook; Lee, Chang Han; Kim, Sera; Kim, Soyoun; Lee, Dong-Ki

doi:10.1007/s10059-011-0160-1

Cited by 7 publications

(8 citation statements)

References 25 publications

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“…Methods for performing these assays are provided in Subheading 3.5 . Genomewide analysis through DNA microarray also revealed that the asiRNA structure can alleviate off-target effects mediated by the seed sequence of the siRNA sense strand ( 22 ) . Methods for assessment of siRNA and asiRNA off-targeting effects by microarray analysis are provided in Subheading 3.6 .…”

Section: Methodsmentioning

confidence: 98%

The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells

Chang

Hong

Dua

et al. 2012

Methods in Molecular Biology

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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.

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Section: Methodsmentioning

confidence: 98%

The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells

Chang

Hong

Dua

et al. 2012

Methods in Molecular Biology

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“…In this study, we developed cp-asiRNA, an RNAi trigger platform with cell membrane-penetrating capability. This RNAi trigger is structurally based on asiRNA with a shortened passenger strand (16 nucleotides), which shows reduced nonspecific effects compared with conventional siRNA (Chang et al, 2009;Hong et al, 2014;Jo et al, 2011). In addition, cp-asiRNA has chemical modifications including cholesterol conjugation, phosphorothioate modification, and 2 0 -O-methyl modification.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we introduced an siRNA structure termed asymmetric siRNA (asiRNA), which had significantly mitigated off-target effects compared with conventional siRNA. (Chang et al, 2009;Hong et al, 2014;Jo et al, 2011) On the basis of the asiRNA backbone structure, we introduced several chemical modifications, such as cholesterol conjugation and phosphorothioate, to enhance the intracellular uptake of the RNAi triggering molecule. We also incorporated 2 0 -O-methyl to enhance the stability of the asiRNA.…”

Section: Introductionmentioning

confidence: 99%

Development of Cell-Penetrating Asymmetric Interfering RNA Targeting Connective Tissue Growth Factor

Hwang¹,

Chang²,

Kim³

et al. 2016

Journal of Investigative Dermatology

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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.

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“…Reflecting the critical roles in cancer, c-Met and its HGF ligand have become leading candidates for targeted cancer therapies (Burgess et al, 2006;Cao et al, 2001;Jo et al, 2011;Kim et al, 2006). However, generation and development of inhibitory antibodies targeting c-Met has been difficult because the divalent structure of antibodies often activates c-Met signaling via receptor dimerization and cross-activation (Ohashi et al, 2000;Prat et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

A New Anti-c-Met Antibody Selected by a Mechanism-Based Dual-Screening Method: Therapeutic Potential in Cancer

Song

Lee

et al. 2012

Molecules and Cells

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-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

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Selection and Optimization of Asymmetric siRNA Targeting the Human c-MET Gene

Cited by 7 publications

References 25 publications

The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells

The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells

Development of Cell-Penetrating Asymmetric Interfering RNA Targeting Connective Tissue Growth Factor

A New Anti-c-Met Antibody Selected by a Mechanism-Based Dual-Screening Method: Therapeutic Potential in Cancer

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