Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

Singh, Saurabh; Narang, Ajit S.; Mahato, Ram I.

doi:10.1007/s11095-011-0608-1

Cited by 182 publications

(140 citation statements)

References 177 publications

(191 reference statements)

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“…They have been used as genetic tools in higher eukaryotes, and are one of the most promising therapeutic modalities for the future. However, delivering these RNAs to specific cells presents a significant challenge that requires traversing the circulatory system while avoiding kidney filtration, degradation by endonucleases, aggregation with serum proteins, and uptake by phagocytes (39). Dose-dependent toxicity has not been definitively determined in mammals.…”

Section: Discussionmentioning

confidence: 99%

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

et al. 2012

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Abstract. The invasive behavior of glioblastoma multiforme (GBM) cells is one of the most important reasons for the poor prognosis of this cancer. For invasion, tumor cells must acquire an ability to digest the extracellular matrix and infiltrate the normal tissue bordering the tumor. Preventing this by altering effector molecules can significantly improve a patient's prognosis. Accumulating evidence suggests that miRNAs are involved in multiple biological functions, including cell invasion, by altering the expression of multiple target genes. The expression levels of miR-218 correlate with the invasive potential of GBM cells. In this study, we found that miR-218 expression was low in glioma tissues, especially in GBM. The data showed an inverse correlation in 60 GBM tissues between the levels of miR-218 and MMP mRNAs (MMP-2, -7 and -9). Additionally, ectopic expression of miR-218 suppressed the invasion of GBM cells whereas inhibition of miR-218 expression enhanced the invasive ability. Numerous members of the MMP family are downstream effectors of the Wnt/LEF1 pathway. Target prediction databases and luciferase data showed that LEF1 is a new direct target of miR-218. Importantly, western blot assays demonstrated that miR-218 can reduce protein levels of LEF1 and MMP-9. We, therefore, hypothesize that miR-218 directly targets LEF1, resulting in reduced synthesis of MMP-9. Results suggest that miR-218 is involved in the invasive behavior of GBM cells and by targeting LEF1 and blocking the invasive axis, miR-218-LEF1-MMPs, it may be useful for developing potential clinical strategies. IntroductionGBM (WHO-grade IV) is the most malignant and frequent brain tumor and has the worst prognosis of any cancer. Despite advances in treatment by surgery combined with radiotherapy or chemotherapy, patients with GBM have a mean survival of only 14.6 months (1). Therefore, new therapeutic targets are urgently needed. A formidable difficulty in treating GBM is that tumor cells diffuse and infiltrate the normal peripheral tissue, therefore it is not possible to completely dissect out and remove the tumor. Degradation of extracellular matrix (ECM) is the defining step in tumor cell invasion and members of the matrix metalloproteinase family (MMPs) have crucial roles in regulating this process (2-4). Thus, understanding and blocking the invasive process may be an effective strategy for treating GBM.The recent discovery of miRNAs is a major advance. Accumulating evidence suggests that miRNAs are involved in multiple biological functions, including cell invasion, by altering the expression of multiple target genes. They bind to the 3' untranslated region (UTR) of target messenger RNAs (mRNAs) to suppress translation or induce degradation of these mRNAs. The roles different miRNAs play have been recently expounded in numerous human tumors as detailed below.Accumulated evidence shows that downregulation of miR-218 can enhance tumor cell invasion and proliferation in several kinds of solid tumors (5,6). In this study, we have identifi...

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Section: Discussionmentioning

confidence: 99%

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

et al. 2012

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“…41 Off-target effects include specific effects, which are caused by the limited degree of complementarity to nontargeted mRNAs, and nonspecific off-target effects, which are due to immune and toxicity-related responses derived from the RNAi construct itself or the delivery vehicle. 42 However, since the induced miR-146a expression in BXSB mice did not exceed the normal range among healthy controls, the specific off-target effects of MS2-miR146a VLP administration were limited. Also, only a slight immune response was observed in MS2-miRNC VLP-treated mice; the acute toxicity of this approach was determined previously.…”

Section: Discussionmentioning

confidence: 99%

MS2 VLP-based delivery of microRNA-146a inhibits autoantibody production in lupus-prone mice

Pan¹,

Jia²,

Zhang³

et al. 2012

IJN

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Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies. Recent studies suggest that microRNAs (miRNAs) play an essential role in immunoregulation and may be involved in the pathogenesis of SLE. Therefore, it was of interest to investigate the potential therapeutic application of miRNAs in SLE, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs) are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR-146a, a well-studied and SLE-related miRNA, in BXSB lupus-prone mice. Methods: VLPs containing miR-146a, and the control VLPs, were prepared using an Escherichia coli expression system and then administered to lupus-prone mice over a 12-day period. We performed an enzyme-linked immunosorbent assay to evaluate the anti-dsDNA antibody, autoantibody to nuclear antigen (ANA), total IgG and total IgM levels in serum. The expression of miR-146a was analyzed by qRT-PCR. SLE-related cytokines as well as some toll-like receptor signaling pathway molecules were also measured. Results: Treatment with MS2-miR146a VLP showed profound effects on lupus-prone BXSB mice, including an increased level of mature miR-146a, which led to a significant reduction in the expression of autoantibodies and total IgG. Remarkably, these mice also exhibited reduced levels of proinflammatorycytokines, including IFN-Interferon-α (IFN-α), and . Moreover, we showed that the toll-like receptor pathway was involved in this regulation. Conclusion: Restoring the loss of miR-146a was effective in eliminating the production of autoantibodies and ameliorating SLE progression in lupus-prone mice. Thus, the induction of dysregulated miRNAs by an MS2 VLP-based delivery system may lead to novel therapies.

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“…Experimental details of each study are described in Table 2. (85) siRNA-mediated downregulation of STAT3, (86) Abundant in Neuroepithelial Area (ANA), (87) Hoxc8, (88) Protein related to DAN and cerberus (PRDC), (89) Noggin, (90,91) Notch, (92) zinc finger Zfp467, (93) guanine nucleotide-binding protein alpha (GNAS1), (94) and prolyl hydroxylase domain-containing protein 2 (PHD2) (94) have been all shown to enhance osteogenic activity of various cell types in vitro. siRNA has been additionally investigated as a supplement to protein delivery, where siRNA against Noggin have been deployed in support of BMP-induced osteogenesis.…”

Section: Bone Induction By Rna Interferencementioning

confidence: 99%

Realizing the potential of gene-based molecular therapies in bone repair

Rose

Uludağ

2013

Journal of Bone and Mineral Research

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A better understanding of osteogenesis at genetic and biochemical levels is yielding new molecular entities that can modulate bone regeneration and potentially act as novel therapies in a clinical setting. These new entities are motivating alternative approaches for bone repair by utilizing DNA-derived expression systems, as well as RNA-based regulatory molecules controlling the fate of cells involved in osteogenesis. These sophisticated mediators of osteogenesis, however, pose unique delivery challenges that are not obvious in deployment of conventional therapeutic agents. Viral and nonviral delivery systems are actively pursued in preclinical animal models to realize the potential of the gene-based medicines. This article will summarize promising bone-inducing molecular agents on the horizon as well as provide a critical review of delivery systems employed for their administration. Special attention was paid to synthetic (nonviral) delivery systems because they are more likely to be adopted for clinical testing because of safety considerations. We present a comparative analysis of dose-response relationships, as well as pharmacokinetic and pharmacodynamic features of various approaches, with the purpose of clearly defining the current frontier in the field. We conclude with the authors' perspective on the future of genebased therapy of bone defects, articulating promising research avenues to advance the field of clinical bone repair. © 2013 American Society for Bone and Mineral Research. KEY WORDS: OSTEOGENESIS; BIOENGINEERING; MOLECULAR PATHWAYS; GENE-BASED THERAPYClinical Need for New Bone-Regeneration Strategies N early 2.2 million bone grafts are performed worldwide annually (1) and up to 20% of fractures are hampered by impaired healing. (2) The economic impact of nonunions is enormous, with the cost of spinal fusions alone reaching $20 billion annually. (3) The gold standard for repair of large segmental defects remains autologous grafts, where bone harvested from a non-weight-bearing site, usually the iliac crest, is used to repair defects. Bone grafts, however, are limited in several aspects, including potency of the grafts and the physiological detriment resulting from harvest surgery. Allografts are alternatively employed, where donor tissue is used to repair the defect, but the risk of disease transmission is always a concern. Allografts are extensively processed to reduce this risk, but osteopotency of the graft could be decreased in this way. (4) Demineralized bone matrix derived from decalcified bone can similarly act as a substitute; its potency, however, is variable and depends on the processing conditions. Synthetic scaffolds have been used to provide a hospitable environment for new bone formation. Scaffolds have been constructed from the organic (collagen) and inorganic (hydroxyapatite [HA]) components of bone, but such scaffolds are incapable of inducing osteogenesis on their own and they are more suitable for smaller defects.Osteogenic proteins are frequently employed to render ost...

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Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

Cited by 182 publications

References 177 publications

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

MS2 VLP-based delivery of microRNA-146a inhibits autoantibody production in lupus-prone mice

Realizing the potential of gene-based molecular therapies in bone repair

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