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

Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease

In vivo silencing of alpha-synuclein using naked siRNA

<p>Abstract</p> <p>Background</p> <p>Overexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease <it>post-mortem</it>. Genetic variability in <it>SNCA </it>contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. <it>SNCA </it>downregulation is therefore a valid therapeutic target for PD.</p> <p>Results</p> <p>We have identified human and murine-specific siRNA molecules which reduce <it>SNCA in vitro</it>. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces <it>SNCA </it>levels. Reduction of <it>SNCA </it>in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion.</p> <p>Conclusion</p> <p>We have developed naked gene-specific siRNAs that silence expression of <it>SNCA in vivo</it>. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of <it>SNCA</it>, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from <it>SNCA </it>overexpression.</p

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20–25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reductionThis work was supported by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed III; by Consellería de Educación e Ordenación Universitaria, Xunta de Galicia's Grupos de referencia competitiva (grant number ED431C 2017/09). The authors thank TÜBİTAK (The Scientific and Technical Research Council of Turkey) for supporting this project (Project number : 217S068). S.A acknowledges the financial support for his postdoctoral research by the 2-INTRATARGET project (PCIN-2017-129/AEI) funded by MINECO-PCIN-2017-129/AEI, under the frame of EuroNanoMed IIIS

Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo siRNA Delivery

Nanoparticles are employed for delivering therapeutics into cells1,2. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell specific internalization, excretion, toxicity, and efficacy3-7. A variety of materials have been explored for delivering small interfering RNAs (siRNAs) - a therapeutic agent that suppresses the expression of targeted genes8,9. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, lack of tissue specificity and potential toxicity10-12. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer targeting ligands (such as peptides and folate) on the nanoparticle surface can be precisely controlled. We show that at least three folate molecules per nanoparticle is required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ∼ 24.2 min) than the parent siRNA (t1/2 ∼ 6 min)

Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters

Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human progesterone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 2′F-RNA, 2′OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications

Sequence-Defined Oligomers from Hydroxyproline Building Blocks for Parallel Synthesis Applications

The functionality of natural biopolymers has inspired significant effort to develop sequence-defined synthetic polymers for applications including molecular recognition, self-assembly, and catalysis. Conjugation of synthetic materials to biomacromolecules has played an increasingly important role in drug delivery and biomaterials. We developed a controlled synthesis of novel oligomers from hydroxyproline-based building blocks and conjugated these materials to siRNA. Hydroxyproline-based monomers enable the incorporation of broad structural diversity into defined polymer chains. Using a perfluorocarbon purification handle, we were able to purify diverse oligomers through a single solid-phase extraction method. The efficiency of synthesis was demonstrated by building 14 unique trimers and 4 hexamers from 6 diverse building blocks. We then adapted this method to the parallel synthesis of hundreds of materials in 96-well plates. This strategy provides a platform for the screening of libraries of modified biomolecules. Keywords: bioconjugates; fluorous synthesis; oligomers; sequence-defined polymers; siRNANational Institutes of Health (U.S.) (Grant EB000244)National Cancer Institute (U.S.) (Grant P30‐CA14051