Recommendations are based on low-quality evidence or on consensus, but are well aligned with recommendations from guidelines from North America. The working groups recommend intensifying research relating to all aspects of management of NP and CR.

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.

Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. In this work, two different diastereoisomers of LNA were studied: the beta-D-LNA and the alpha-L-LNA (abbreviated as beta-D-LNA and alpha-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing beta-D-LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA-DNA-LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif-dependent, and requires the right balance between gap size and affinity. Compared to beta-D-LNA, alpha-L-LNA shows superior stability against a 3'-exonuclease. The design possibilities of alpha-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of alpha-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, alpha-L-LNA and beta-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with alpha-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with beta-D-LNA results in a drop in antisense potency. Our findings indicate that alpha-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3′-to-5′ exoribonuclease and the 2′-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication–transcription complex and virus replication.