The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson–Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.

Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell–impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.

Background MicroRNA-129-1 (miR-129-1) seems to behave as a tumour suppressor since its decreased expression is associated with different tumours such as glioblastoma multiforme (GBM). GBM is the most common form of brain tumours originating from glial cells. The impact of miR-129-1 downregulation on GBM pathogenesis has yet to be elucidated. Methods MiR-129-1 was overexpressed in GBM cells, and its effect on proliferation was investigated by cell cycle assay. MiR-129-1 predicted targets (CDK6, IGF1, HDAC2, IGF2BP3 and MAPK1) were also evaluated by western blot and luciferase assay. Results Restoration of miR-129-1 reduced cell proliferation and induced G1 accumulation, significantly. Several functional assays confirmed IGF2BP3, MAPK1 and CDK6 as targets of miR-129-1. Despite the fact that IGF1 expression can be suppressed by miR-129-1, through 3′-untranslated region complementary sequence, we could not find any association between IGF1 expression and GBM. MiR-129-1 expression inversely correlates with CDK6, IGF2BP3 and MAPK1 in primary clinical samples. Conclusion This is the first study to propose miR129-1 as a negative regulator of IGF2BP3 and MAPK1 and also a cell cycle arrest inducer in GBM cells. Our data suggests miR-129-1 as a potential tumour suppressor and presents a rationale for the use of miR-129-1 as a novel strategy to improve treatment response in GBM.