MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. For example, miR-33a and miR-33b have a crucial role in controlling cholesterol and lipid metabolism in concert with their host genes, the sterol-regulatory element-binding protein (SREBP) transcription factors. Other metabolic miRNAs, such as miR-103 and miR-107, regulate insulin and glucose homeostasis, whereas miRNAs such as miR-34a are emerging as key regulators of hepatic lipid homeostasis. The discovery of circulating miRNAs has highlighted their potential as both endocrine signalling molecules and disease markers. Dysregulation of miRNAs may contribute to metabolic abnormalities, suggesting that miRNAs may potentially serve as therapeutic targets for ameliorating cardiometabolic disorders.

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MicroRNAs in metabolism and metabolic disorders.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo. Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.

Therapeutic targeting of microRNAs: current status and future challenges

The recent discovery of new potent therapeutic molecules that do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). CPPs were first discovered based on the potency of several proteins to enter cells. Numerous CPPs have been described so far, which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization and the second involving formation of stable, non-covalent complexes with drugs. Nowadays, CPPs constitute very promising tools for non-invasive cellular import of cargo and have been successfully applied for in vitro and in vivo delivery of therapeutic molecules varying from small chemical molecule, nucleic acids, proteins, peptides, liposomes and particles. This review will focus on the structure/function and cellular uptake mechanism of CPPs in the general context of drug delivery. We will also highlight the application of peptide carriers for the delivery of therapeutic molecules and provide an update of their clinical evaluation.

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Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics

Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches - including chemical modification, bioconjugation and the use of nanocarriers - which aim to address the delivery challenge.

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Advances in oligonucleotide drug delivery

動注化学療法における pharmacokinetics と臨床

Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

/pdf/targeted-inhibition-of-mirna-maturation-with-morpholinos-2hwgxr2g5y.pdf

Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development

Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy.

Antisense molecules do not readily cross cell membranes. This has limited the use of antisense to systems where techniques have been worked out to introduce the molecules into cells, such as embryos and cell cultures. Uncharged antisense bearing a group of guanidinium moieties on either a linear peptide or dendrimer scaffold can enter cells by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. These technologies allow systemic administration of antisense, making gene knockdowns and splice modification feasible in adult animals; this review presents examples of such animal studies. Techniques developed with PPMOs, which are an arginine-rich cell-penetrating peptide linked to a Morpholino oligo, can also be performed using commercially available Vivo-Morpholinos, which are eight guanidinium groups on a dendrimeric scaffold linked to a Morpholino oligo. Antisense-based techniques such as blocking translation, modifying pre-mRNA splicing, inhibiting miRNA maturation and inhibiting viral replication can be conveniently applied in adult animals by injecting PPMOs or Vivo-Morpholinos.

https://www.mdpi.com/1420-3049/14/3/1304/pdf

Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos

Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and maturation of miRNA, although they can also inhibit other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.

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Using Morpholinos to control gene expression.

Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice. Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previous assay techniques produced confounding artifacts that led to the old non-endocytotic, membrane-penetrating peptide model. The artifacts result from fixing cells for fluorescent microscopy and from using non-trypsinized cells for flow cytometry. Fixing cells redistributes the peptide or peptide-oligo conjugates associated with the outside of cell membranes and trapped in endosomes, giving apparent diffuse cytosolic and nuclear fluorescence. Cationic peptides bound to the outer surface of cells, if not removed, skew fluorescence data obtained by flow cytometry, leading to the earlier conclusions. Upregulation assays now provide a tool for comparing the efficacy of conjugates, measuring oligo uptake by quantitating antisense activity of conjugates. These assays, developed in cell culture and mouse models, are faster and have higher signal-to-noise ratios than downregulation assays. Thus, a convenient and effective method now exists to screen transport peptides.

Jon D. Moulton

Papers

Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development

Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy.

Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos

Using Morpholinos to control gene expression.

Peptide-assisted delivery of steric-blocking antisense oligomers.