Efficient Tandem Cu-Catalyzed Click Synthesis of Multi-Sugar-Modified Oligonucleotides.

Nucleic acids in the form of siRNA, antisense oligonucleotides or mRNA are currently explored as new promising modalities in the pharmaceutical industry. Particularly, the success of mRNA-vaccines against SARS-CoV-2, along with the successful development of the first sugar-modified siRNA therapeutics has inspired the field. The development of nucleic acid therapeutics requires efficient chemistry to link oligonucleotides to chemical structures that can improve stability, boost cellular uptake, or enable specific targeting. For the siRNA therapeutics currently in use, modification of the 3'-end of the oligonucleotides with triple-N-acetylgalactosamine (GalNAc)3 was shown to be of significance. This modification is currently achieved via a cumbersome multi-step synthesis and subsequent loading onto the solid support material. Here, we report the development of a bifunctional click-reactive linker that allows the modification of oligonucleotides in a tandem click reaction with multiple sugars, regardless of the position within the oligonucleotide, with remarkable efficiency and in a one-pot reaction.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis.

BACKGROUND & AIMS: Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. METHODS: Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. RESULTS: Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. CONCLUSION: Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. IMPACT AND IMPLICATIONS: Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.