Potential of application of the RNA interference phenomenon in the treatment of new coronavirus infection COVID-19

COVID-19 has killed more than 4 million people to date and is the most significant global health problem. The first recorded case of COVID-19 had been noted in Wuhan, China in December 2019, and already on March 11, 2020, World Health Organization declared a pandemic due to the rapid spread of this infection. In addition to the damage to the respiratory system, SARS-CoV-2 is capable of causing severe complications that can affect almost all organ systems. Due to the insufficient effectiveness of the COVID-19 therapy, there is an urgent need to develop effective specific medicines. Among the known approaches to the creation of antiviral drugs, a very promising direction is the development of drugs whose action is mediated by the mechanism of RNA interference (RNAi). A small interfering RNA (siRNA) molecule suppresses the expression of a target gene in this regulatory pathway. The phenomenon of RNAi makes it possible to quickly create a whole series of highly effective antiviral drugs, if the matrix RNA (mRNA) sequence of the target viral protein is known. This review examines the possibility of clinical application of siRNAs aimed at suppressing reproduction of the SARS-CoV-2, taking into account the experience of similar studies using SARS-CoV and MERS-CoV infection models. It is important to remember that the effectiveness of siRNA molecules targeting viral genes may decrease due to the formation of viral resistance. In this regard, the design of siRNAs targeting the cellular factors necessary for the reproduction of SARS-CoV-2 deserves special attention.

Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP).

Physicochemical properties of lipopeptide-based liposomes and their complexes with siRNA

siRNA/cationic liposome complexes are efficient systems for transmembrane delivery. The aim of this study was to prepare a novel complex consisted of lipotripeptide OrnOrnGlu(C16H33)2 and siRNA molecule and examined their physicochemical properties. Electron microscopy study has shown that the siRNA/liposome complex (m/m 1/10) tends to form sandwich-like structures that may protect nucleic acid from nuclease degradation. Photon correlation spectroscopy data indicate that the particle size increased after siRNA adding, but did not exceed 300 nm in diameter, while z-potential of lipoplexes decreased from 22 mV to 14 mV, compared to the empty liposomes thus indicating positive charge neutralization by negatively charged siRNA. These data allow to hypothesize that such size and total positive charge could provide efficient cellular uptake by endocytosis. That may have good prospects for gene silencing therapy.

Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

Single molecule force spectroscopy has been applied to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface.

Synthesis, physico-chemical and biological properties of DNA and RNA oligonucleotides containing short alkylamino internucleotide bond

The condensation of the 5′-O-DMT-3′-deoxy-3′-aminothymidine with 3′-O-TBDMS-thymidine- 5′-aldehyde, followed by reduction of the resultant imine derivative and removal of tert-butyldimethylsilyl (TBDMS) protecting group, provided a dimer (denoted as TNHT), which is a congener of dithymidine phosphate with the phosphate linkage 3′-O-P(O)(OH)-O-5′ replaced with an amino group (–NH–). After phosphitylation of the 3′-OH group, the dimer TNHT was introduced (by the standard phosphoramidite approach) into a central part of the nonadecathymidylate. This oligomer exhibited lower affinity to the complementary single and double stranded DNA complements as compared to unmodified T19 oligonucleotide. The cleavage of modified oligomer with the snake venom and calf spleen phosphodiesterases was completely suppressed at the site of modification. RNA oligomers containing the TNHT dimer were used for preparation of siRNA molecules directed towards mRNA of BACE1 (beta-site amyloid precursor protein cleaving enzyme). The presence of the TNHT units at the 3′-ends of the RNA strands of the siRNA molecule (the siRNA itself is an effective gene expression inhibitor for BACE1) preserved the gene silencing activity and improved the stability of the modified siRNA in 10% fetal bovine serum.

Vascular Endothelial Growth Factor (VEGF) Is a Critical Autocrine Survival Factor in Acute Promyelocytic Leukemia (APL).

We have previously demonstrated (Kini AR et. al. Blood.2001;97:3919–24) that VEGF is expressed in APL and is regulated by all-trans retinoic acid (ATRA). In this study, we examined the role of VEGF as a autocrine survival factor in APL, by using siRNA to VEGF in cultured NB4 APL cells. The siRNA molecules were transfected into the APL cells by electroporation and resulted in almost complete (>95%) knock-down of VEGF expression. The VEGF knock-down resulted in significant apoptosis (>50%) within 96 hours. To verify the specificity of the VEGF siRNA, we used a siRNA molecule with a single nucleotide mismatch mutation, as well as a scrambled siRNA molecule. These control siRNA molecules did not result in VEGF knock-down, nor did they cause apoptosis, confirming the specificity of the VEGF siRNA and its effect. In contrast to the striking induction of apoptosis by VEGF knock-down, external blocking of VEGF and VEGF receptors by addition of neutralizing antibodies did not cause apoptosis. In addition, the concentration of VEGF within the cells was over 100-fold higher than the external VEGF concentration. These results suggest that VEGF mediates its actions through an internal autocrine mechanism. Intriguingly, immunfluorescence studies showed that VEGF is strongly expressed in the nucleus, suggesting a novel nuclear role for VEGF. To study the possible role of the VEGF receptors in internal loops we knocked-down the VEGF receptors using siRNA molecules. VEGF receptor knock-down did not affect survival of the APL cells. To summarize, we demonstrate that VEGF is a survival factor for APL cells, and acts independent of the VEGF receptors through an internal autocrine mechanism. These results indicate that targeting internal VEGF may be an effective therapeutic strategy for APL.