Low-level shRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice

Beer, Shelly; Bellovin, David I.; Lee, Joyce; Komatsubara, Kimberly M.; Wang, Lora S.; Koh, Huishan; Börner, Kathleen; Storm, Theresa A.; Davis, Corrine R.; Kay, Mark A.; Felsher, Dean W.; Grimm, Dirk

doi:10.1038/mt.2009.222

Cited by 40 publications

(34 citation statements)

References 46 publications

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“…Mechanistically, it is likely that Ago-1 to -4 cosaturation can further perturb miRNA function and/or cause global changes in the transcriptome. The latter was indeed observed in cells depleted of individual Ago proteins (32), and altered miRNA expression and activity were also among our most striking phenotypes in shRNA-expressing cells and mice (3,4).…”

Section: Discussionmentioning

confidence: 72%

“…We had indeed noted substantial changes in hepatic miRNA levels in livers of shRNAtreated ailing mice (3,4), and miRNA activities were consistently disturbed in shRNA-transfected cells (3,4,6,12). The fact that such adverse effects on miRNAs are seen with transfected siRNAs as well (12)(13)(14)(15)(16) implies that one or more key factors in the RNAi pathway are rate-limiting in cell culture.…”

Section: Introductionmentioning

confidence: 98%

“…However, while emerging data suggest RNAi treatment to be effective and well tolerated and thus spur tremendous optimism (2), there are also escalating somber reports in animals of severe adverse side effects from in vivo RNAi. These include our early findings of substantial hepatotoxicities and/or fatalities in adult mice following hepatic high-level expression of more than 50 distinct shRNAs, delivered by a potent adenoassociated viral (AAV) vector (3,4). Similarly, Davidson's group noted striatal neurotoxicities and cerebellar Purkinje cell loss in mice treated with shRNAs against the huntingtin (5) and SCA1 (6) genes, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

Grimm¹,

Wang²,

Lee³

et al. 2010

J. Clin. Invest.

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174

203

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shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short-and long-term clinical gene silencing in humans.

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Section: Discussionmentioning

confidence: 72%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

Grimm¹,

Wang²,

Lee³

et al. 2010

J. Clin. Invest.

Self Cite

174

203

View full text Add to dashboard Cite

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“…Moreover, several reports showed cytotoxic effects induced by shRNA. [127][128][129][130] Therefore, artificial microRNA seem to be the preferred tool to knock down a target gene because they permit inducible or tissue specific expression from polymerase II promoters and have not been associated with any cytotoxic effects to date. 129,131,132 Such a platelet specific approach, however, has currently not been reported on.…”

Section: Transplantation Modelsmentioning

confidence: 99%

Model systems of genetically modified platelets

Thijs

Deckmyn

Broos

2012

Blood

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Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed antithrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet- specific IntroductionBlood platelets play part in a myriad of processes, such as inflammation, tumor growth and metastasis, immunology and, of course, thrombosis and blood clotting where they provide a first and crucial line of defense against vascular injury, thus maintaining normal hemostasis. 1,2 Primary hemostasis starts when platelets recognize a site of vascular injury where the subendothelial matrix is exposed, bind to collagen, and become activated. 3 The subsequent rise in intracellular calcium triggers conformational changes in integrin receptors, degranulation, exposition of a procoagulant surface, and generation and release of secondary agonists resulting in a thrombus that will cover the site of injury and prevent further blood loss. 4 Platelets are furthermore an important factor in thrombotic events, such as stroke and myocardial infarction. 5 To identify more proteins regulating platelet function that may serve as new targets for the development of anti-thrombotics or in the prevention of bleeding, the platelet research community has seen the completion of several large-scale screening programs and the spectacular rise in the "platelet-omics" field. Several genome-wide association studies and subsequent meta-analysis in patients with coronary artery disease and healthy volunteers identified numerous genetic loci that are possibly involved in regulating platelet formation, count, volume, and function and might confer a risk for coronary artery disease. [6][7][8][9][10][11] On the other hand, gene expression profiling of healthy volunteer platelets, in combination with comparative microarray analysis between in vitro differentiated megakaryocytes (MKs) and closely related cell types, established a comprehensive platelet transcriptome. 6,[12][13][14][15][16][17][18][19] Finally, advanced proteomics studies identified proteins of the platelet sheddome, secretome, interactome, kinome, and phosphoproteome potentially involved in platelet function. 20 The overall result is a large number of newly identified gene products for which we are only beginning to understand their ...

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“…Artificial siRNA/shRNA molecules can target unwanted mRNAs (offtargeting) 31 or induce immunological responses that cause toxicity. [32][33][34] High level shRNA expression can cause morbidity and mortality in mouse models, 35,36 but these adverse effects are usually dose-dependent. These results suggest that one or more key components of the RNAi pathway in mammalian cells may become saturated.…”

Section: Rnai Strategiesmentioning

confidence: 99%

Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors

Herrera-Carrillo

Liu

Berkhout

2017

Human Gene Therapy Methods

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The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed. These miRNAs are based on the molecules that induce the natural RNAi pathway in mammals and humans: the endogenously expressed miRNAs. Significant efforts focused on the construction and delivery of miRNA cassettes in order to solve basic biology questions or to design new therapy strategies. Several viral vectors have been developed, which are particularly useful for the delivery of miRNA expression cassettes to specific target cells. Each vector system has its own unique set of distinct properties. Thus, depending on the specific application, a particular vector may be most suitable. This field was previously reviewed for different viral vector systems, and now the recent progress in the field of miRNA-based gene-silencing approaches using lentiviral vectors is reported. The focus is on the unique properties and respective limitations of the available vector systems for miRNA delivery.Keywords: miRNA cassettes, gene therapy, viral vectors, vector tropism INTRODUCTION NON-CODING RNA MOLECULES play an essential role in gene regulation of the cell via a mechanism called RNA interference (RNAi). The RNAi mechanism is evolutionary conserved in eukaryotes and triggers the sequence-specific inhibition or degradation of a single or a unique set of complementary mRNAs. Three small RNA classes have been described to participate in mammalian RNAi mechanisms: microRNAs (miRNAs), 1,2 endogenous small interfering RNAs (endo-siRNAs), 3 and PIWI-associated RNAs (piRNAs). 4 The endo-siRNAs and piRNAs are involved in suppression of transposable elements. The miRNAs are broadly involved in the regulated expression of multiple cellular genes and execute their effect at the posttranscriptional level. 5,6 MiRNA-mediated regulation of gene expression plays a significant role in cell metabolism and cellular developmental and differentiation processes in mammals. More than a thousand human miRNAs have already been identified that are involved in the regulated expression of around 30% of all genes, which is a conservative estimate.7 Because of the miRNA abundance and their important regulatory functions, these molecules have received much attention from the scientific community over the last decade. For instance, efforts have been made to explain the biological function of the natural miRNAs by identifying the target mRNA and the role in cellular physiology. On the other hand, the design of man-made miRNA mimics to impose control over gene expression beca...

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Low-level shRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice

Cited by 40 publications

References 46 publications

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

Model systems of genetically modified platelets

Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors

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