Sirna research paper

Bar-Sagi has published over peer-reviewed articles in leading scientific journals. His main research interests include the molecular mechanisms of mammalian cell-cycle control and responses to DNA damage, and the cancer-predisposing aberrations of these regulatory pathways. Jiri Bartek has a total of more than publications in peer reviewed journals about in Nature, Science and Cellwith over He is currently member of the editorial boards of 10 high-medium impact biomedical journals and has won a number of awards including:

Sirna research paper

Abstract siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects.

Here, we Sirna research paper the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 PLK1 and kinesin spindle protein KSP in mice.

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This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis.

Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

Since the characterization of Sirna research paper fundamental gene-silencing mechanism, tremendous progress has been made in developing siRNA as a potentially novel class of therapeutic agent for a broad spectrum of diseases including cancer, viral infection, and metabolic disorders.

Many siRNA targets in oncology have been described in the literature, although direct evidence that their therapeutic effects in tumor models are mediated by RNAi is notably lacking.

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The interpretation of antitumor activity attributable to siRNAs is problematic due to the potential for off-target effects of the nucleic acids, including their propensity to activate immune responses through TLR-dependent 2 — 4 and TLR-independent mechanisms 56.

These types of response are known to elicit antitumor effects, primarily through the actions of IFNs and inflammatory cytokines that exert antiangiogenic, proapoptotic, and adjuvant effects that enhance cellular immunity 78.

Sirna research paper

Induction of the innate immune response by nucleic acids can also have significant toxicologic consequences reviewed in ref. MacLachlan, unpublished observations has shown that human subjects can be exquisitely sensitive to the toxic effects of these agents when compared with preclinical models.

Therefore additional caution is required if considering an immune stimulatory siRNA for clinical development 14 The incorporation of modified nucleotide chemistries into siRNA has been widely utilized to improve their pharmacologic and nuclease-resistant properties We first reported that extensive chemical modification to siRNA molecules could provide the additional benefit of preventing their recognition by the mammalian immune system To establish proof that systemically administered siRNAs can elicit RNAi-mediated anticancer efficacy in the absence of measurable immune activation, we selected the essential cell-cycle proteins kinesin spindle protein KSP, also referred to as Eg5 19 and polo-like kinase 1 PLK1 20 as validated cancer targets with well-characterized mechanisms of direct tumor cell killing.

KSP is a mitotic spindle motor protein that drives chromosome segregation during mitosis. Inhibition of KSP blocks the formation of bipolar mitotic spindles, causing cell-cycle arrest, activation of the mitotic checkpoint, and induction of apoptosis In mammalian cells, PLK1 acts to phosphorylate a number of cell-cycle proteins including Cdc25C, cyclin B, cohesin subunit SCC-1, subunits of the anaphase promoting complex, mammalian kinesin-like protein 1, and other kinesin related proteins.

This diverse array of substrates reflects the multiple roles of PLK1 in mitosis and cytokinesis Overexpression of PLK1, observed in many human tumor types, is a negative prognosticator of patient outcome reviewed in ref.

Depletion of PLK1 may also sensitize cancer cells to the proapoptotic activity of small-molecule drugs 25likely due to the role of PLK1 in the DNA damage and spindle assembly checkpoints.

One of the primary barriers to realizing the potential of siRNA therapeutics is the requirement for drug-delivery vehicles to facilitate disease site targeting, cellular uptake, and cytoplasmic delivery of the siRNA 26 — Common approaches to delivery include complexing the siRNA with polycations such as polyethyleneimine 2930 and cyclodextrin polymers 31 as well as incorporation into cationic lipid—based carriers 171826 We have previously described the development of stable nucleic acid lipid particles SNALP as an effective systemic delivery vehicle for targeting siRNAs to the murine and nonhuman primate liver and have demonstrated therapeutic effects in silencing endogenous hepatocyte 1826 and viral gene transcripts The accumulation of SNALP within tissues of clinical interest takes advantage of passive disease-site targeting 3334whereby charge-neutral carriers of suitable size around nm diameter or smaller can pass through the fenestrated epithelium of tumors, sites of inflammation, and the healthy liver.

Sirna research paper

This avoids the requirement for active targeting moieties such as peptides, antibodies, and receptor ligands that may otherwise be candidates for incorporation into siRNA-delivery vehicles to enhance target-cell selectivity 3135 Results demonstrate that rationally designed siRNAs targeting PLK1 or KSP, when delivered with an effective systemic delivery vehicle, are able to affect therapeutic gene silencing in solid tumors.

The specificity and mechanism of action is confirmed using a combination of methodologies that demonstrate RNAi-mediated silencing of target mRNA causing mitotic disruption in tumor cells typical of target inhibition.

This can be achieved in the complete absence of immune stimulation through the use of appropriately designed, chemically modified siRNAs. PLK1 represents a validated gene target in oncology whose inhibition is known to cause mitotic arrest and apoptosis in proliferating tumor-cell cultures We designed and screened a panel of PLK1 siRNA for antiproliferative activity in the human HT29 colon cancer cell line Supplemental Figure 1; supplemental material available online with this article; doi: Duplicate plates were assessed for cell viability at 72 hours.

E Decreased cell viability is associated with the induction of apoptosis. Prior to the in vivo assessment of synthetic siRNA, it is essential to anticipate the potential effects of immune stimulation on the biological system under consideration and take steps to mitigate this risk We regarded this step as a prerequisite to conducting in vivo studies in order to conclude the specificity of antitumor effects that may be observed.

JCI - Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice

In this case, each combination of the 2 modified sense and AS strands generated a duplex with potency equivalent to that of the native KSP sequence, confirming preservation of RNAi activity Figure 2 E. We believe that this siRNA design and screening approach can be applied to any given sequence to rapidly identify siRNAs in which the chemical modifications are well tolerated with respect to RNAi activity and predicted to fully abrogate immune stimulation.This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.

RNAi induction using siRNAs or their biosynthetic precursors Transfection of an exogenous siRNA can be problematic, since the gene knockdown effect is . the siRNA groups 72 h and 96 h after transfection, while there were no significant differences between the NC and control group after 24 h and 48 h after transfection.

Sirna Research Paper

Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction.

However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. If the siRNA effector is delivered to the cell it will “activate” RISC, resulting in potent and specific silencing of the targeted mRNA.

Because of the potency and selectivity of RNAi, it has become the methodology of choice for silencing specific gene expression in mammalian cells. Sirna Research Paper increase and effects off-target reduce to Algorithm Design siRNA Rosetta the using libraries siRNA and siRNA predesigned for synthesis quality and design Advanced.

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RNAi mechanisms and applications