Trans-amplifying RNA expressing functional miRNA mediates target gene suppression and simultaneous transgene expression.

The co-delivery of microRNAs (miRNAs) and protein-coding RNA presents an opportunity for a combined approach to gene expression and gene regulation for therapeutic applications. Protein delivery is established using long mRNA, self-, and trans-amplifying RNA (taRNA), whereas miRNA delivery typically uses short synthetic oligonucleotides rather than incorporating it as a precursor into long RNA. Although miRNA delivery into the cell cytoplasm using long genomes of RNA viruses has been described, concerns have remained regarding low processing efficiency. However, miRNA precursors can be released from long cytoplasmic alphaviral RNA by a cytoplasmic fraction of Drosha. taRNA, a promising vector platform for infectious disease vaccination, uses a nonreplicating mRNA expressing an alphaviral replicase to amplify a protein-coding short transreplicon-RNA (STR) in trans. To investigate the possibility of simultaneously delivering protein expression and gene silencing, we tested whether a taRNA system can carry and release functional miRNA to target cells. Here, we show that mature miRNA is released from STRs and silences specific targets in a replication-dependent manner for several days without compromising the expression of STR-encoded proteins. Our findings suggest that incorporating miRNAs into the taRNA vector platform has the potential for gene regulation alongside the expression of therapeutic genes.

Prediction of tumor-specific splicing from somatic mutations as a source of neoantigen candidates.

Motivation: Neoantigens are promising targets for cancer immunotherapies and might arise from alternative splicing. However, detecting tumor-specific splicing is challenging because many non-canonical splice junctions identified in tumors also appear in healthy tissues. To increase tumor-specificity, we focused on splicing caused by somatic mutations as a source for neoantigen candidates in individual patients. Results: We developed the tool splice2neo with multiple functionalities to integrate predicted splice effects from somatic mutations with splice junctions detected in tumor RNA-seq and to annotate the resulting transcript and peptide sequences. Additionally, we provide the tool EasyQuant for targeted RNA-seq read mapping to candidate splice junctions. Using a stringent detection rule, we predicted 1.7 splice junctions per patient as splice targets with a false discovery rate below 5% in a melanoma cohort. We confirmed tumor-specificity using independent, healthy tissue samples. Furthermore, using tumor-derived RNA, we confirmed individual exon-skipping events experimentally. Most target splice junctions encoded neoepitope candidates with predicted major histocompatibility complex (MHC)-I or MHC-II binding. Compared to neoepitope candidates from non-synonymous point mutations, the splicing-derived MHC-I neoepitope candidates had lower self-similarity to corresponding wild-type peptides. In conclusion, we demonstrate that identifying mutation-derived, tumor-specific splice junctions can lead to additional neoantigen candidates to expand the target repertoire for cancer immunotherapies. Availability and implementation: The R package splice2neo and the python package EasyQuant are available at https://github.com/TRON-Bioinformatics/splice2neo and https://github.com/TRON-Bioinformatics/easyquant, respectively.

Clinical Determinants of Thrombin Generation Measured in Presence and Absence of Platelets-Results from the Gutenberg Health Study.

The tendency of a plasma sample to generate thrombin, a central enzyme in blood coagulation, might be an important indicator of prothrombotic risk linked to cardiovascular disease (CVD), but the presence of platelets may be a critical determinant. Clinical data, laboratory markers and thrombin generation (TG), investigated in both platelet-rich plasma (PRP) and platelet-free plasma (PFP) at 1 pM TF, were available in 407 individuals from the Gutenberg Health Study. Given the well-known effect of anticoagulants on TG, subjects taking anticoagulants (n = 15) have been excluded resulting in 392 subjects for further analysis. Lag time, endogenous thrombin potential (ETP) and peak height were the investigated parameters of a TG curve. Multivariable linear regression analysis was used to identify TG determinants. Mean platelet volume (MPV) and platelet count were both negatively associated to lag time and positively to peak height (MPV, ß:6.35 [2.66; 10.0]; platelet count, ß:0.111 [0.054; 0.169]) in PRP only. C-reactive protein was positively associated with lag time and ETP in both PRP and PFP, with a stronger effect on ETP in PRP (PRP, ß:76.7 [47.5; 106]; PFP, ß:34.8 [10.3; 59.2]). After adjustment for fibrinogen, the relation between CRP and ETP was attenuated in PRP and PFP. Of the traditional cardiovascular risk factors (CVRFs), obesity was positively associated to TG in PRP only. Our findings support that TG, particularly in PRP, relates to traditional CVRFs in a representative sample from a population-based study. Assessment of procoagulant activity in a platelet-dependent manner by TG is a promising tool for assessing individual risk for CVD.