Chemical Modification Of siRNA Research Articles

A Convenient Method for the Synthesis of 2′‐O‐Cyanoethylated Nucleotides and Their Application in the Solid‐Phase Synthesis of Related RNA Analogs

AbstractAlthough small interfering RNA (siRNA) is a key player among gene inhibition therapeutics, there are many obstacles to the development of siRNA drugs due to inherent properties of oligonucleotides, including the unsatisfactory stability of unmodified siRNA, poor pharmacokinetic distribution, and the toxicity induced by off‐target effects. To maximize treatment potency, chemical modification of siRNA has undoubtedly been the most successful strategy by far. Widely applied modifications include phosphorothioate linkages, 2′‐O‐methyl modifications, and 2′‐fluoro modifications, among others. To extend the family of chemical modifications for oligonucleotides, 2′‐O‐cyanoethylated RNA analogs were developed through the replacement of the 2′‐hydroxyl group with a 2′‐O‐cyanoethyl group (‐OCH2CH2CN). This modification can provide several advantages over unmodified RNA, such as increased stability, improved binding affinity to complementary DNA or RNA strands, and resistance to degradation by cellular nucleases. The 2′‐O‐cyanoethyl‐modified RNAs not only are applied in RNA silencing machinery but also act as research tools for studying RNA structure and function or for developing RNA‐based diagnostics. Therefore, the efficient synthesis, deprotection, purification, and characterization of 2′‐O‐cyanoethylated RNAs deserves more attention. This protocol describes the chemical synthesis of 2′‐O‐cyanoethylated nucleotides and the solid‐phase synthesis, deprotection, and purification of 2′‐O‐cyanoethylated RNAs. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Preparation of 6‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl adenosine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 2: Preparation of 4‐N‐acetyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl cytidine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 3: Preparation of 2‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl guanine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 4: Preparation of 5′‐O‐dimethoxytrityl‐2′‐O‐2‐cyanoethyl uridine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 5: Solid‐phase synthesis of 2′‐O‐cyanoethylated RNA analogsBasic Protocol 6: Deprotection and purification of synthesized 2′‐O‐cyanoethyl‐RNAs

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 reduction.

Small interfering RNA: Discovery, pharmacology and clinical development-An introductory review.

Post-transcriptional gene silencing targets and degrades mRNA transcripts, silencing the expression of specific genes. RNA interference technology, using synthetic structurally well-defined short double-stranded RNA (small interfering RNA [siRNA]), has advanced rapidly in recent years. This introductory review describes the utility of siRNA, by exploring the underpinning biology, pharmacology, recent advances and clinical developments, alongside potential limitations and ongoing challenges. Mediated by the RNA-induced silencing complex, siRNAs bind to specific complementary mRNAs, which are subsequently degraded. siRNA therapy offers advantages over other therapeutic approaches, including ability of specifically designed siRNAs to potentially target any mRNA and improved patient adherence through infrequent administration associated with a very long duration of action. Key pharmacokinetic and pharmacodynamic challenges include targeted administration, poor tissue penetration, nuclease inactivation, rapid renal elimination, immune activation and off-target effects. These have been overcome by chemical modification of siRNA and/or by utilising a range of delivery systems, increasing bioavailability and stability toallow successful clinical translation. Patisiran (hereditary transthyretin-mediated amyloidosis) was the first licensed siRNA, followed by givosiran (acute hepatic porphyria), lumasiran (primary hyperoxaluria type 1) and inclisiran (familial hypercholesterolaemia), which all use N-acetylgalactosamine (GalNAc) linkage for effective liver-directed delivery. Others are currently under development for indications varying from rare genetic diseases to common chronic non-communicable diseases (hypertension, cancer). Technological advances are paving the way for broader clinical use. Ongoing challenges remain in targeting organs beyond the liver and reaching special sites (e.g., brain). By overcoming these barriers, siRNA therapy has the potential to substantially widen its therapeutic impact.

Ideal and Reality: Barricade in the Delivery of Small Interfering RNA for Cancer Therapy.

Small interfering RNA (siRNA) has been demonstrated to be a powerful tool for silencing post-transcriptional gene expression, and thus exhibits great potential in cancer gene therapy. However, siRNA technology has not become an established strategy for cancer treatment, as it is a considerable challenge to deliver siRNA to the targeting sites. In this review, barricades in the systemic delivery of siRNA were systematically introduced, from pharmacokinetics, biodistribution and intracellular behavior. Meanwhile, current and potential strategies for overcoming the barricades were elucidated, including chemical modification of siRNA, nanomaterial-based delivery systems, and targeting and stimuli-responsive smart carriers. In conclusion, the siRNA-based gene silencing drug will gain an important position for treating human diseases if we can solve the gap between ideal and reality.

High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

Use of Self-Delivery siRNAs to Inhibit Gene Expression in an Organotypic Pachyonychia Congenita Model

Although RNA interference offers therapeutic potential for treating skin disorders, delivery hurdles have hampered clinical translation. We have recently demonstrated that high pressure, resulting from intradermal injection of large liquid volumes, facilitated nucleic acid uptake by keratinocytes in mouse skin. Furthermore, similar intradermal injections of small interfering RNA (siRNA; TD101) into pachyonychia congenita (PC) patient foot lesions resulted in improvement. Unfortunately, the intense pain associated with hypodermic needle administration to PC lesions precludes this as a viable delivery option for this disorder. To investigate siRNA uptake by keratinocytes, an organotypic epidermal model, in which pre-existing endogenous gene or reporter gene expression can be readily monitored, was used to evaluate the effectiveness of "self-delivery" siRNA (i.e., siRNA chemically modified to enhance cellular uptake). In this model system, self-delivery siRNA treatment resulted in reduction of pre-existing fluorescent reporter gene expression under conditions in which unmodified controls had little or no effect. Additionally, treatment of PC epidermal equivalents with self-delivery "TD101" siRNA resulted in marked reduction of mutant keratin 6a mRNA with little or no effect on wild-type expression. These results indicate that chemical modification of siRNA may overcome certain limitations to transdermal delivery (specifically keratinocyte uptake) and may have clinical utility for inhibition of gene expression in the skin.

Effective Stabilization and Delivery of siRNA: Reversible siRNA−Phospholipid Conjugate in Nanosized Mixed Polymeric Micelles

siRNA is a powerful tool to control cellular processes at the post-transcriptional level. However, its therapeutic potential is limited because of low stability in biological fluids and the lack of simple and efficient delivery systems. Chemical modification of siRNA could be used to increase its intracellular delivery, but may affect its specific activity. To overcome these obstacles, we suggest a simple and effective system capable of stabilization, delivery, and subsequent release of free active siRNA within cells. With this in mind, we reversibly modified the double-stranded GFP-siRNA with a phosphothioethanol (PE) portion via the reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate in nanosized PEG-PE micelles. In the mixed siRNA-S-S-PE/PEG-PE micelles obtained, siRNA was well-protected against degradation by nucleases for at least 24 h, and was released easily from these nanoparticles in free form in the presence of glutathione (GSH) at a concentration mimicking the intracellular levels. In GFP-C166 endothelial cells, mixed GFP-siRNA-S-S-PE/PEG-PE micelles down-regulate the GFP production 50-fold more effectively than free siRNA. In addition, siRNA-containing micelles showed none of the cytotoxic side effects typical for siRNA delivery systems that are based on electrostatic association of siRNA with cationic carriers. Thus, a reversible siRNA-phospholipid conjugate formulated into mixed micelles with PEG-PE can be an effective, nontoxic system for stabilization and delivery of siRNA.

In vivo efficacy and off-target effects of locked nucleic acid (LNA) and unlocked nucleic acid (UNA) modified siRNA and small internally segmented interfering RNA (sisiRNA) in mice bearing human tumor xenografts.

The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.

SiRNA Delivery Using Nanocarriers – An Efficient Tool for Gene Silencing

Small interfering RNAs (siRNA) are one of the most recent additions used to silence gene expression. At present siRNA is the most extensively used gene-silencing technique over other nucleic-acid based approaches to treat disease including cancer, hepatitis, respiratory disease, cardiovascular diseases, neuronal disease and autoimmune disease. However, systemic delivery of siRNA in vivo, remains to be the biggest challenge to be overcome. Various strategies have been developed to deliver siRNA efficiently into target cell such as chemical modification of siRNA, physical strategies, viral and non viral-vectors mediated delivery. Among all the approaches non viral vectors including lipoplexes and polyplexes were found to be most successful which have been reviewed in this article. Further therapeutic applications of RNAi have also been briefly reviewed.

Astrocytes are very sensitive to develop innate immune responses to lipid‐carried short interfering RNA

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll-like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells. We examined the effects of various siRNA sequences prepared with lipids (oligofectamine). Lipid-siRNA induced variable degrees of silencing-independent nonspecific effects, e.g. increased Stat1 and Cox-2 expression and release of IL-6 and IP-10 in primary astroglia. This was prevented through chemical modification of siRNA by nucleoside 2'-O-methylation, without impairing specific gene silencing. Lipid-siRNA also induced nonspecific responses in purified astroglia, but not in microglia, or 3T3 cells. The highest TLR7 and TLR3 mRNA expression was found in microglia and purified astroglia, respectively. Accordingly, the TLR3 agonist poly(I:C) (PIC) induced higher release of IFN-beta in primary and purified astroglia than in microglia. As siRNA, PIC induced IP-10, Stat1, VCAM-1, and Cox-2 and increased TLR3 mRNA expression. The effects of lipid-siRNA in purified astrocytes were attenuated after silencing TLR3 or TLR7 expression, and by the PKR inhibitor 2-aminopurine. Furthermore, lipid-siRNA induced the expression of RIG-I. In contrast, siRNA devoid of lipids did not enter the astrocytes, did not silence gene expression, and did not induce Stat1 or Cox-2. The results show that, in astroglia, lipid-siRNA induces innate immune responses that are mediated, at least in part, by intracellular mechanism dependent on TLR7, TLR3, and helicases.

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA–protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176–183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 (‘slicer’ enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson–Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (TM) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

Potential Applications of RNA Interference-Based Therapeutics in the Treatment of Cardiovascular Disease

RNA interference (RNAi) in eukaryotes is a recently identified phenomenon in which small double stranded RNA molecules called short interfering RNA (siRNA) interact with messenger RNA (mRNA) containing homologous sequences in a sequence-specific manner. Ultimately, this interaction results in degradation of the target mRNA. Because of the high sequence specificity of the RNAi process, and the apparently ubiquitous expression of the endogenous protein components necessary for RNAi, there appears to be little limitation to the genes that can be targeted for silencing by RNAi. Thus, RNAi has enormous potential, both as a research tool and as a mode of therapy. Several recent patents have described advances in RNAi technology that are likely to lead to new treatments for cardiovascular disease. These patents have described methods for increased delivery of siRNA to cardiovascular target tissues, chemical modifications of siRNA that improve their pharmacokinetic characteristics, and expression vectors capable of expressing RNAi effectors in situ. Though RNAi has only recently been demonstrated to occur in mammalian tissues, work has advanced rapidly in the development of RNAi-based therapeutics. Recently, therapeutic silencing of apoliporotein B, the ligand for the low density lipoprotein receptor, has been demonstrated in adult mice by systemic administration of chemically modified siRNA. This demonstrates the potential for RNAi-based therapeutics, and suggests that the future for RNAi in the treatment of cardiovascular disease is bright.

RNA Interference with Chemically Modified siRNA

siRNA has become an indispensable tool for functional characterization of genes. It has also demonstrated tremendous potential as a new generation of drug candidates. Although the technology works very well to a great panel of cells in vitro, it is still a challenge to translate the success into in vivo target validation easiness and, even more difficult, into therapeutic applications. With a number of chemically modified compounds under initial clinical trial from several commercial entities, the interests in chemical modification of siRNA have become heightened. In this review we have tried to touch on most of the chemical modifications of RNA that have been tested in the siRNA landscape, but maintained a focus on the backbone modifications, and 2'-modifications on the ribose ring. It is anticipated that more modifications and more systematic comparisons between different modifications will be performed to draw more educated conclusions over some of the modifications.

Targeted Delivery of siRNA

Therapeutic application of siRNA requires delivery to the correct intracellular location, to interact with the RNAi machinery within the target cell, within the target tissue responsible for the pathology. Each of these levels of targeting poses a significant barrier. To overcome these barriers several strategies have been developed, such as chemical modifications of siRNA, viral nucleic acid delivery systems, and nonviral nucleic acid delivery systems. Here, we discuss progress that has been made to improve targeted delivery of siRNA in vivo for each of these strategies.

Chemical modifications of siRNA for improved functionality

Chemical modifications of siRNA for improved functionality

Effects on RNA interference in gene expression (RNAi) in cultured mammalian cells of mismatches and the introduction of chemical modifications at the 3'-ends of siRNAs.

The highly specific posttranscriptional silencing of gene expression induced by double-stranded RNA (dsRNA) is known as RNA interference (RNAi) and has been demonstrated in plants, nematodes, Drosophila, and protozoa, as well as in mammalian cells. The suppression of expression of specific genes by chemically synthesized 21-nucleotide (21-nt) RNA duplexes has been achieved in various lines of mammalian cells, and this technique might prove to be a valuable tool in efforts to analyze biologic functions of genes in mammalian cells. In order to investigate the utility of potential modifications that can be introduced into small interfering RNAs (siRNAs) and also to study their functional anatomy, we synthesized different types of siRNA targeted to mRNA of Jun dimerization protein 2 (JDP2). Our detailed analysis demonstrated that siRNAs with only one mismatch, relative to the target, on the antisense strand had reduced RNAi effect, whereas the corresponding mutation on the sense strand did not interfere with the RNAi. Moreover, one 2-hydroxyethylphosphate (hp) substitution at the 3'-end of the antisense strand but not of the sense strand also prevented RNAi, whereas a related modification at the 3'-end of either strand, using 2'-O,4'-C-ethylene thymidine (eT), which is a component of ethylene-bridge nucleic acids (ENA), completely abolished RNAi. These results support the hypothesis that the two strands have different functions in RNAi in cultured mammalian cells and indicate that their chemical modification of siRNAs at the 3'-end of the sense strand exclusively is possible, without loss of RNAi activity, depending on the type of modification. Because modification at the 3'-end of the antisense strand by hp or eT abolished the RNAi effect, it appears possible that the 3'-end is recognized by the RNA-induced silencing complex (RISC).