The Roles of Individual Mammalian Argonautes in RNA Interference In Vivo

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.Alnylam Pharmaceuticals (Firm

Investigation of barriers to non-viral gene delivery and design of novel polymer-based gene delivery systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2003.Includes bibliographical references.The safe and effective delivery of therapeutic genes is the most significant challenge facing gene therapy today. Viral vectors remain the dominant approach for addressing the delivery problem; however, concerns regarding the safety of viral vectors have resulted in an increasing interest in non-viral vectors. Non-viral vectors offer the promise of improved safety, but because they have yet to match the functional sophistication of viral vectors, their transfection efficiencies have lagged those of viral vectors. The rational design of functional non-viral vectors requires a thorough understanding of both the cell's sophisticated machinery and the vector material's functional properties. We have developed a novel, flow cytometry based tool for investigating both the cellular uptake and lysosomal trafficking of non-viral vectors, two important barriers to efficient gene transfer. Using this and other tools we investigated the gene transfer properties of polyethylenimine (PEI), a highly effective non-viral vector material. We demonstrated that the transfection efficiency of this polymer is due to its ability to avoid lysosomal degradation as a result of its buffering capacity, providing quantitative validation of the proton sponge hypothesis. By studying the gene transfer properties of a library of polymeric vectors, we were able to elucidate new vector structure-function relationships. We also investigated the combined impact of non-structural factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on gene transfer. The findings of these studies have lead to the development of non-viral vectors with transfection efficiencies surpassing those of PEI and Lipofectamine 2000, two of the best commercially available non-viral vectors.by Akin Akinc.Ph.D

Shielding of Lipid Nanoparticles for siRNA Delivery: Impact on Physicochemical Properties, Cytokine Induction, and Efficacy

Formulation of short interfering RNA (siRNA) into multicomponent lipid nanoparticles (LNP) is an effective strategy for hepatic delivery and therapeutic gene silencing. This study systematically evaluated the effect of polyethylene glycol (PEG) density on LNP physicochemical properties, innate immune response stimulation, and in vivo efficacy. Increased PEG density not only shielded LNP surface charge but also reduced hemolytic activity, suggesting the formation of a steric barrier. In addition, increasing the PEG density reduced LNP immunostimulatory potential as reflected in cytokine induction both in vivo and in vitro. Higher PEG density also hindered in vivo efficacy, presumably due to reduced association with apolipoprotein E (ApoE), a protein which serves as an endogenous targeting ligand to hepatocytes. This effect could be overcome by incorporating an exogenous targeting ligand into the highly shielded LNPs, thereby circumventing the requirement for ApoE association. Therefore, these studies provide useful information for the rational design of LNP-based siRNA delivery systems with an optimal safety and efficacy profile

RNAi-nanoparticulate manipulation of gene expression as a new functional genomics tool in the liver

BACKGROUND and AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver

siRNAs targeting CDS and 3′UTR differ in their Ago2 dependence <i>in vivo</i>.

<p>Data points are levels of mRNA measured by qRT-PCR in the livers (harvested 24 hours post-injection) of mice i.v.-injected with LNP-formulated siRNAs, as described in Materials and Methods, expressed as a percentage of mRNA levels in control siRNA-treated animals. (A) mRNA expression of Argonautes and Dicer1 in Ago2^fl/fl and Ago2^−/− mouse liver (mean ± s.d., n = 4). (B) Levels of Fads1 mRNA in livers of Ago2^−/− mice and Ago2^fl/fl mice (mean ± s.d., n = 2–3) treated with siRNAs targeting Fads1 CDS or 3′UTR at 0.5 mg/kg. (C) Rab5c mRNA levels after treatment with siRNAs targeting its CDS or 3′UTR-far at 1 mg/kg (mean ± s.d., n = 2–3). (D) FVII mRNA in the liver of animals treated with siRNAs (mean ± s.d., n = 4–5, doses: 0.8 mg/kg for siRNA targeting CDS and 0.4 mg/kg for siRNA targeting 3′UTR) and FVII protein in the serum quantified by chromogenic assay. (E) Activity of siRNAs targeting CDS and 3′UTR in the absence of Ago1, 3, or 4. Legend in the bottom left corner of the graph also indicates the line in the X-axis describing the treatment type by gene (mean ± s.d., n = 3). Mice knockout for different individual Argonaute genes and C57BL/6 control animals were i.v.-injected with one of two combinations of LNP-formulated siRNAs targeting Fads1 and Rab5c (CDS-targeting for one gene, at 0.8 mg/kg and 3′UTR-targeting for the other gene, at 0.4 mg/kg) or with control Luciferase siRNA at 1.2 mg/kg.</p