Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3′-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5′-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.     

Reductively responsive siRNA-conjugated hydrogel nanoparticles for gene silencing

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as particle replication in nonwetting templates (PRINT), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during postfabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the pro-drug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.     

Photoregulation of Gene Expression with Amantadine-Modified Caged siRNAs through Host–Guest Interactions

RNA interference is an essential and powerful tool for targeting and verifying specific gene functions. Conditional control of small interfering RNA (siRNA) activity, especially using light activation, is a potential method for regulating target gene expression and functions. In this study, a series of photolabile siRNAs with amantadine modification have been rationally designed and developed through host–guest interactions between amantadine and β-cyclodextrin derivatives to enhance the blocking effect of siRNA binding and/or RNA-induced silencing complex processing. These caged siRNAs with amantadine modification at the 5′ end of antisense-strand RNA were efficiently inactivated through the host–guest interactions between amantadine and β-cyclodextrin. Photomodulation of the gene silencing activity of these amantadine-modified caged siRNAs targeting both exogenous and endogenous genes was successfully achieved, which indicates that host–guest interactions could be a new strategy for developing new caged siRNAs for gene photoregulation with low leaking activity.     

Dissecting RNA-interference pathway with small molecules

RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that occurred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a decrease in target RNA cleavage by activated RNA-induced silencing complex (RISC*). Interestingly, when activated RISC*, which harbors unwound antisense siRNA, was treated with ATPA-18 in vitro, target RNA cleavage was not affected, indicating that this compound inhibited siRNA unwinding or steps upstream of unwinding in the RNAi pathway. Our results also establish the timing of siRNA unwinding and show that siRNA helicase activity is required for RNAi. ATPA-18 analogs will therefore provide a new class of small molecules for studying RNAi mechanisms in a variety of model organisms and deciphering in vivo genetic functions through reverse genetics.     

PAMAM Dendrimers for siRNA Delivery: Computational and Experimental Insights

Short double‐stranded RNAs, which are known as short interfering RNA (siRNA), can be used to specifically down‐regulate the expression of the targeted gene in a process known as RNA interference (RNAi). However, the success of gene silencing applications based on the use of synthetic siRNA critically depends on efficient intracellular delivery. Polycationic branched macromolecules such as poly(amidoamine) (PAMAM) dendrimers show a strong binding affinity for RNA molecules and, hence, can provide an effective, reproducible, and relatively nontoxic method for transferring siRNAs into animal cells. Notwithstanding these perspectives, relatively few attempts have been made so far along these lines to study in detail the molecular mechanisms underlying the complexation process between PAMAMs and siRNAs. In this work we combine molecular simulation and experimental approaches to study the molecular requirements of the interaction of RNA‐based therapeutics and PAMAM dendrimers of different generations. The dendrimers and their siRNA complexes were structurally characterized, and the free energy of binding between each dendrimer and a model siRNA was quantified by using the well‐known MM/PBSA approach. DOSY NMR experiments confirmed the structural in silico prediction and yielded further information on both the complex structure and stoichiometry at low N/P ratio values. siRNA/PAMAM complex formation was monitored at different N/P ratios using gel retardation assays, and a simple model was proposed, which related the amount of siRNA complexed to the entropy variation upon complex formation obtained from the computer simulations.     

In situ electroporation of surface-bound siRNAs in microwell arrays

Gene silencing using RNA interference (RNAi) has become a prominent biological tool for gene annotation, pathway analysis, and target discovery in mammalian cells. High-throughput screens conducted using whole-genome siRNA libraries have uncovered rich sets of new genes involved in a variety of biological processes and cellular models of disease. However, high-throughput RNAi screening is not yet a mainstream tool in life science research because current screening platforms are expensive and onerous. Miniaturizing the RNAi screening platform to reduce cost and increase throughput will enable its widespread use and harness its potential for rapid genome annotation. With this aim, we have combined semi-conductor microfabrication and nanolitre dispensing techniques to develop miniaturized electroporation-ready microwell arrays loaded with siRNA molecules in which multiplexed gene knockdown can be achieved. Arrays of microwells are created using high-aspect ratio biocompatible photoresists on optically transparent and conductive Indium-Tin Oxide (ITO) substrates with integrated micro-electrodes to enable in situ electroporation. Non-contact inkjet microarraying allows precise dispensing of nanolitre volumes into the microwell structures. We have achieved parallel electroporation of multiple mammalian cells cultured in these microwell arrays and observed efficient knockdown of genes with surface-bound, printed siRNAs. Further integration of microfabrication and non-contact nanolitre dispensing techniques described here may enable single-substrate whole-genome siRNA screening in mammalian cells.     

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles

In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of −13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.     

In silico molecular docking analysis of the human Argonaute 2 PAZ domain reveals insights into RNA interference

RNA interference (RNAi) is a critical cellular pathway activated by double stranded RNA and regulates the gene expression of target mRNA. During RNAi, the 3′ end of siRNA binds with the PAZ domain, followed by release and rebinding in a cyclic manner, which deemed essential for proper gene silencing. Recently, we provided the forces underlying the recognition of small interfering RNA by PAZ in a computational study based on the structure of Drosophila Argonaute 2 (Ago2) PAZ domain. We have now reanalyzed these data within the view of the new available structures from human Argonauts. While the parameters of weak binding are correlated with higher (RNAi) in the Drosophila model, a different profile is predicted with the human Ago2 PAZ domain. On the basis of the human Ago2 PAZ models, the indicators of stronger binding as the total binding energy and the free energy were associated with better RNAi efficacy. This discrepancy might be attributable to differences in the binding site topology and the difference in the conformation of the bound nucleotides.     

Potential Use of Dendrimer/α-Cyclodextrin Conjugate as a Novel Carrier for Small Interfering RNA (siRNA)

RNA interference (RNAi) is the mechanism of gene silencing-mediated messenger RNA degradation by small interference RNA (siRNA), which becomes a powerful tool for genetic analysis and novel gene therapy. However, one of the major obstacles for siRNA delivery is the difficulty to cross the biological membrane due to its hydrophilicity and high molecular weight. We evaluated the potential use of the starburst polyamidoamine dendrimer (generation 3) conjugate with α-cyclodextrin (α-CyD) having an average degree of substitution of 2.4 (α-CDE conjugate) as a siRNA carrier for RNAi. The ternary complex composed of pGL2 control vector (pDNA)/pGL2 siRNA/α-CDE conjugate showed higher pGL2 siRNA sequence-specific gene silencing effects without off-target effects than those of commercial transfection reagents such as Lipofectamine™2000 (LP), TransFast™ (TF) and Lipofectin™ (LF). These results suggest that α-CDE conjugate has the potential to be a novel carrier for siRNA.     

Localized, targeted, and sustained siRNA delivery

Short interfering RNA (siRNA) functions directly in the cytoplasm, where it is assembled into an RNA-induced silencing complex (RISC). The localized delivery of siRNA to a specific site in vivo is highly challenging. There are many disease states in which a systemic effect of RNAi may be desirable; some examples include non-localized cancers, HIV, neurodegenerative diseases, respiratory viruses, and heart and vascular disease. In this Concept, we will focus on the localized delivery of siRNA to a target site using various delivery modalities. In certain tissues, such as the eye, central nervous system and lung, it has been demonstrated that a simple injection of naked siRNA will silence gene expression specifically in that tissue. To achieve local gene silencing in other tissues, a variety of approaches have been pursued to help stabilize the siRNA and facilitate uptake; they include chemical modification of the siRNA or complexation within liposomes or polymers to form nanoparticles. Recently, the use of macroscopic biomaterial scaffolds for siRNA delivery has been reported, and although there is still significant work to be done in this area to optimize the delivery systems, it is an important area of research that offers the potential for having great impact on the field of siRNA delivery.     

Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin E Modification

Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5′ terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6‐fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE‐modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE‐binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

Ultra‐pH‐Responsive and Tumor‐Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti‐Cancer Efficacy

RNA interference (RNAi) gene silencing technologies have shown significant potential for treating various diseases, including cancer. However, clinical success in cancer therapy remains elusive, mainly owing to suboptimal in vivo delivery of RNAi therapeutics such as small interference RNA (siRNA) to tumors. Herein, we developed a library of polymers that respond to a narrow pH change (ultra‐pH‐responsive), and demonstrated the utility of these materials in targeted and deep tumor‐penetrating nanoparticle (NP) for in vivo RNAi. The new NP platform is mainly composed of the following key components: i) internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration; ii) polyethylene glycol (PEG) chains to prolong blood circulation; and iii) sharp pH‐responsive hydrophobic polymer to improve endosome escape. Through systematic studies of structure–function relationship, the optimized RNAi NPs (<70 nm) showed efficient gene silencing and significant inhibition of tumor growth with negligible toxicities in vivo.     

Synthesis of Site‐Specifically Phosphate‐Caged siRNAs and Evaluation of Their RNAi Activity and Stability

A complete set of new photolabile nucleoside phosphoramidites were synthesized, then site‐specifically incorporated into sense or antisense strands of siRNA for phosphate caging. Single caging modification was made along siRNA strands and their photomodulation of gene silencing were examined by using the firefly luciferase reporter gene. Several key phosphate positions were then identified. Furthermore, multiple caging modifications at these key positions led to significantly enhanced photomodulation of gene silencing activity, suggesting a synergistic effect. The caging group on both the terminally phosphate‐caged siRNA and the single‐stranded caged RNA has comparatively high stability, whereas hydrolysis of the caged group from the internally caged siRNA was observed, irrespective of the presence of Mg²⁺. Molecular dynamic simulations demonstrated that enhanced hydrolysis of the caging group on internally phosphate‐caged siRNAs was due to easy fragmentation of the caging group upon formation of the pentavalent intermediate of the phosphotriester with attack by water. The caging group in the terminally phosphate‐caged siRNA or single‐stranded caged RNA prefers to form π–π stacks with nearby nucleobases. In addition to providing explanations for previous observations, this study sheds further light on the design of caged oligonucleotides and indicates the direction of future development of nucleic acid drugs with phosphate modifications.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.