Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

γ Peptide Nucleic Acid-Based miR-122 Inhibition Rescues Vascular Endothelial Dysfunction in Mice Fed a High-Fat Diet.

The blood levels of microRNA-122 (miR-122) is associated with the severity of cardiovascular disorders, and targeting it with efficient and safer miR inhibitors could be a promising approach. Here, we report the generation of a γ-peptide nucleic acid (γPNA)-based miR-122 inhibitor (γP-122-I) that rescues vascular endothelial dysfunction in mice fed a high-fat diet. We synthesized diethylene glycol-containing γP-122-I and found that its systemic administration counteracted high-fat diet (HFD)-feeding-associated increase in blood and aortic miR-122 levels, impaired endothelial function, and reduced glycemic control. A comprehensive safety analysis established that γP-122-I affects neither the complete blood count nor biochemical tests of liver and kidney functions during acute exposure. In addition, long-term exposure to γP-122-I did not change the overall adiposity, or histology of the kidney, liver, and heart. Thus, γP-122-I rescues endothelial dysfunction without any evidence of toxicity in vivo and demonstrates the suitability of γPNA technology in generating efficient and safer miR inhibitors.

Induction of splice correction by cell-penetrating peptide nucleic acids.

BACKGROUND: Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing. METHODS: HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs. RESULTS: All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis. CONCLUSIONS: These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.