MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

https://www.gene-quantification.de/kruetzfeld-antagomirs-2005.pdf

Silencing of microRNAs in vivo with ‘antagomirs’

In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.

Knocking down barriers: advances in siRNA delivery

Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients. Long, double-stranded RNAs were first shown to mediate RNAi in Caenorhabditis elegans, and the potential use of RNAi for human therapy has been demonstrated by the finding that small interfering RNAs (siRNAs; approximately 21-base-pair double-stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response. We are at present conducting the first in-human phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumour biopsies from melanoma patients obtained after treatment show the presence of intracellularly localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is, to our knowledge, a first for systemically delivered nanoparticles of any kind). Furthermore, a reduction was found in both the specific messenger RNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) levels when compared to pre-dosing tissue. Most notably, we detect the presence of an mRNA fragment that demonstrates that siRNA-mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. Together, these data demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action.

/pdf/evidence-of-rnai-in-humans-from-systemically-administered-3pba6uirdb.pdf

Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications.

/pdf/gold-nanoparticles-for-biology-and-medicine-409qkznwvh.pdf

Gold nanoparticles for biology and medicine.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

In vivo genome editing using Staphylococcus aureus Cas9

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as noncanonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9–12nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potencyenhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

/pdf/modulation-of-thermal-stability-can-enhance-the-potency-of-3jnz5nuls9.pdf

Gary Lavine

Papers

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

Modulation of thermal stability can enhance the potency of siRNA

Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes.

An automated algorithm for sequence confirmation of chemically modified oligonucleotides by tandem mass spectrometry.