Dramatic advances in understanding of the roles RNA plays in normal health and disease have greatly expanded over the past 10 years and have made it clear that scientists are only beginning to ...comprehend the biology of RNAs. It is likely that RNA will become an increasingly important target for therapeutic intervention; therefore, it is important to develop strategies for therapeutically modulating RNA function. Antisense oligonucleotides are perhaps the most direct therapeutic strategy to approach RNA. Antisense oligonucleotides are designed to bind to the target RNA by well-characterized Watson-Crick base pairing, and once bound to the target RNA, modulate its function through a variety of postbinding events. This review focuses on the molecular mechanisms by which antisense oligonucleotides can be designed to modulate RNA function in mammalian cells and how synthetic oligonucleotides behave in the body.
Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic ...acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.
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► Single-stranded RNA functions through RNAi in cells ► Single-stranded RNA achieves allele-selective inhibition of huntingtin ► Allele-selective inhibition proceeds through a miRNA-like mechanism ► Direct ssRNA infusion reduces mutant huntingtin levels in mouse brain.
Chemically stabilized single-stranded RNAs function within the RISC complex to silence mutant huntingtin expression in mouse brain. Allele-selective silencing represents a new therapeutic avenue for modulating Huntington disease.
Abstract
The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 ...activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.
Graphical Abstract
Graphical Abstract
Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.
The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler ...path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5′ phosphate to achieve activity in vivo, and we developed a metabolically stable 5′-(E)-vinylphosphonate (5′-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.
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► Chemically modified single-stranded siRNAs silence gene expression in animals ► ss-siRNAs require 5′ phosphate and association with AGO2 for gene silencing ► Passenger strand is dispensable for potent RNAi-mediated gene silencing ► Single-stranded siRNAs provide an alternate option for RNAi therapeutics
Chemically modified RNA oligonucleotides load into Ago2-containing RISC complexes to effect RNA silencing without the need for a passenger strand. These single-stranded siRNAs can be applied directly in animals and thus offer new alternatives for developing therapeutics and diagnostics.
Abstract
Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized ...that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.
Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene ...disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.
Abstract
We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance ...therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.
Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense ...oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ∼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.
Abstract
Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 ...RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.
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