Current Development of siRNA Bioconjugates: From Research to the Clinic

Small interfering RNAs (siRNAs) acting via RNA interference mechanisms are able to recognize a homologous mRNA sequence in the cell and induce its degradation. The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degradation of siRNAs by nucleases in biological fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo (e.g., viruses, cationic lipids, polymers, nanoparticles), but all have limitations for therapeutic use. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i.e., the covalent connection of siRNAs with biogenic molecules (lipophilic molecules, antibodies, aptamers, ligands, peptides, or polymers). Bioconjugates are “ideal nanoparticles” since they do not need a positive charge to form complexes, are less toxic, and are less effectively recognized by components of the immune system because of their small size. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing

Activation of Innate Immunity by Therapeutic Nucleic Acids

Nucleic acid-based therapeutics have gained increased attention during recent decades because of their wide range of application prospects. Immunostimulatory nucleic acids represent a promising class of potential drugs for the treatment of tumoral and viral diseases due to their low toxicity and stimulation of the body’s own innate immunity by acting on the natural mechanisms of its activation. The repertoire of nucleic acids that directly interact with the components of the immune system is expanding with the improvement of both analytical methods and methods for the synthesis of nucleic acids and their derivatives. Despite the obvious progress in this area, the problem of delivering therapeutic acids to target cells as well as the unresolved issue of achieving a specific therapeutic effect based on activating the mechanism of interferon and anti-inflammatory cytokine synthesis. Minimizing the undesirable effects of excessive secretion of inflammatory cytokines remains an unsolved task. This review examines recent data on the types of immunostimulatory nucleic acids, the receptors interacting with them, and the mechanisms of immunity activation under the action of these molecules. Finally, data on immunostimulatory nucleic acids in ongoing and completed clinical trials will be summarized

Structural Modifications of siRNA Improve Its Performance In Vivo

The use of small interfering RNA (siRNA) in the clinic gives a wide range of possibilities for the treatment of previously incurable diseases. However, the main limitation for biomedical applications is their delivery to target cells and organs. Currently, delivery of siRNA to liver cells is a solved problem due to the bioconjugation of siRNA with N-acetylgalactosamine; other organs remain challenging for siRNA delivery to them. Despite the important role of the ligand in the composition of the bioconjugate, the structure and molecular weight of siRNA also play an important role in the delivery of siRNA. The basic principle is that siRNAs with smaller molecular weights are more efficient at entering cells, whereas siRNAs with larger molecular weights have advantages at the organism level. Here we review the relationships between siRNA structure and its biodistribution and activity to find new strategies for improving siRNA performance

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3′ end of the sense strand of siRNA and cholesterol on the silencing activity of “light” and “heavy” modified siRNAs. All 3′-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5′-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3′-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy

Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene

Chemical modifications are an effective way to improve the therapeutic properties of small interfering RNAs (siRNAs), making them more resistant to degradation in serum and ensuring their delivery to target cells and tissues. Here, we studied the carrier-free biodistribution and biological activity of a nuclease-resistant anti-MDR1 cholesterol-siRNA conjugate in healthy and tumor-bearing severe combined immune deficiency (SCID) mice. The attachment of cholesterol to siRNA provided its efficient accumulation in the liver and in tumors, and reduced its retention in the kidneys after intravenous and intraperitoneal injection. The major part of cholesterol-siRNA after intramuscular and subcutaneous injections remained in the injection place. Confocal microscopy data demonstrated that cholesterol-siRNA spread deep in the tissue and was present in the cytoplasm of almost all the liver and tumor cells. The reduction of P-glycoprotein level in human KB-8-5 xenograft overexpressing the MDR1 gene by 60% was observed at days 5–6 after injection. Then, its initial level recovered by the eighth day. The data showed that, regardless of the mode of administration (intravenous, intraperitoneal, or peritumoral), cholesterol-siMDR efficiently reduced the P-glycoprotein level in tumors. The designed anti-MDR1 conjugate has potential as an adjuvant therapeutic for the reversal of multiple drug resistance of cancer cells. Keywords: cholesterol-containing siRNA, MDR1, 2’-O-methyl modification, tumor xenograft in SCID mice, multiple drug resistance, biodistribution of siRN

Influence of the Composition of Cationic Liposomes on the Performance of Cargo Immunostimulatory RNA

In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, β, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or β, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect

Trimeric Small Interfering RNAs and Their Cholesterol-Containing Conjugates Exhibit Improved Accumulation in Tumors, but Dramatically Reduced Silencing Activity

Cholesterol derivatives of nuclease-resistant, anti-MDR1 small-interfering RNAs were designed to contain a 2’-OMe-modified 21-bp siRNA and a 63-bp TsiRNA in order to investigate their accumulation and silencing activity in vitro and in vivo. The results showed that increasing the length of the RNA duplex in such a conjugate increases its biological activity when delivered using a transfection agent. However, the efficiency of accumulation in human drug-resistant KB-8-5 cells during delivery in vitro in a carrier-free mode was reduced as well as efficiency of target gene silencing. TsiRNAs demonstrated a similar biodistribution in KB-8-5 xenograft tumor-bearing SCID mice with more efficient accumulation in organs and tumors than cholesterol-conjugated canonical siRNAs; however, this accumulation did not provide a silencing effect. The lack of correlation between the accumulation in the organ and the silencing activity of cholesterol conjugates of siRNAs of different lengths can be attributed to the fact that trimeric Ch-TsiRNA lags mainly in the intercellular space and does not penetrate sufficiently into the cytoplasm of the cell. Increased accumulation in the organs and in the tumor, by itself, shows that using siRNA with increased molecular weight is an effective approach to control biodistribution and delivery to the target organ