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Development of Long Asymmetric siRNA Structure for Target Gene Silencing and Immune Stimulation in Mammalian Cells.

Post-transcriptional regulation of transcript abundances by RNA interference (RNAi) is a widely conserved regulatory mechanism to control cellular processes. We previously introduced an alternative siRNA structure called asymmetric siRNA (asiRNA), and showed that asiRNA exhibits comparable gene-silencing efficiency with reduced off-target effects compared with conventional siRNAs. However, to what extent the length of the guide strand affects the gene-silencing efficiency of asiRNAs is still elusive. In this study, we analyzed in detail the gene-silencing ability of asiRNAs along the guide strand length and immunostimulatory capacity of asiRNAs. We generated asiRNAs containing various guide strand lengths ranging from 25 to 29 nt, called long asiRNA (lasiRNA). We found that the gene-silencing activity of lasiRNAs decreased as the length of the guide strand increased. Nonetheless, the 3'-end overhangs that are complementary to the target gene have higher efficiency for gene silencing compared with mismatched overhangs. In addition, we found that the silencing efficiency of lasiRNAs correlates with their Ago2-binding affinity. Finally, replacing the mismatched overhang with a TLR7- or TLR9-associated immune response motif induced a toll-like receptor (TLR)-specific immune response and retained gene-silencing activity. Our findings demonstrate that lasiRNA structures can be tailored to function as bifunctional siRNA, which trigger a specific immune response combined with target gene silencing. Taken together, we anticipate that our findings provide a road map for the subsequent development of immune-stimulating lasiRNA, which bear the potential to be applied for therapeutic benefits.

Delivery Characterization of SPL84 Inhaled Antisense Oligonucleotide Drug for 3849 + 10 kb C- > T Cystic Fibrosis Patients.

Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led to increased numbers of ASO regulatory approvals. In this study, we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease cystic fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when targeting the cell nucleus. The distribution of SPL84 throughout the lungs, penetration into the epithelial cells and nucleus, and structural stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of our inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL84 through hyperconcentrated mucus was also demonstrated. Our results, supported by a promising preclinical pharmacological effect of full restoration of cystic fibrosis transmembrane conductance regulator channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients. In addition, successfully tackling the lung distribution of SPL84 offers immense opportunities for further development of SpliSense's inhaled ASO-based drugs for unmet needs in pulmonary diseases.

New Oligonucleotide 2'-O-Alkyl N3'→P5' (Thio)-Phosphoramidates as Potent Antisense Agents: Physicochemical Properties and Biological Activity.

We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'→P5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.

Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics.

Preclinical evaluation of nucleic acid therapeutics (NATs) in relevant experimental model systems is essential for NAT drug development. As part of COST Action "DARTER" (Delivery of Antisense RNA ThERapeutics), a network of researchers in the field of RNA therapeutics, we have conducted a survey on the experimental model systems routinely used by our members in preclinical NAT development. The questionnaire focused on both cellular and animal models. Our survey results suggest that skin fibroblast cultures derived from patients is the most commonly used cellular model, while induced pluripotent stem cell-derived models are also highly reported, highlighting the increasing potential of this technology. Splice-switching antisense oligonucleotide is the most frequently investigated RNA molecule, followed by small interfering RNA. Animal models are less prevalent but also widely used among groups in the network, with transgenic mouse models ranking the top. Concerning the research fields represented in our survey, the mostly studied disease area is neuromuscular disorders, followed by neurometabolic diseases and cancers. Brain, skeletal muscle, heart, and liver are the top four tissues of interest reported. We expect that this snapshot of the current preclinical models will facilitate decision making and the share of resources between academics and industry worldwide to facilitate the development of NATs.

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