Biology subjects

Seephetdee, C.

Publications and source records attributed to Seephetdee, C..

4 recordsLinked to original sources

bioRxiv2026

Efficient permuted intron-exon RNA circularization system based on the LtrB group II Intron from Lactococcus lactis

Circular RNAs (circRNAs) have emerged as promising platforms for stable RNA expression and therapeutic applications, but methods for their generation remain limited. Here, we engineered the Lactococcus lactis LtrB group II intron into a permuted intron-exon (PIE) configuration to enable efficient circRNA formation (circLtrB). Among the tested split designs, LtrB domain IV cleavage yielded the highest circularization efficiency. Although LtrB encodes LtrA maturase required for efficient in vivo splicing, in vitro application of LtrA to the PIE constructs resulted in lower circularization efficiency than self-splicing conditions. We further examined circularization under distinct salt conditions and showed that NH4Cl- and KCl-based splicing buffers independently support high circularization efficiencies (>80%), while producing distinct splicing intermediates, indicating ion-specific mechanistic effects on circularization. Extending outer homology arms did not enhance circularization efficiency, demonstrating that efficient circRNA formation in the circLtrB platform is independent of extended exon pairing interactions. Using this system, we generated circRNAs across a range of coding sequence lengths, highlighting its payload flexibility. To optimize translation, we evaluated multiple internal ribosome entry sites (IRES), including Aichivirus, Crohivirus, CVB3, improved EMCV, HRV, and Salivirus, and found that the CVB3 IRES drove the highest translation efficiency. Linear NLuc showed faster decay kinetics, while circLtrB-NLuc exhibited more sustained expression over time. Transcriptome-wide RNA sequencing revealed comparable innate immune responses between circular and linear RNAs. Together, these results establish circLtrB as a versatile and efficient platform for circRNA production, providing mechanistic insight into group II intron-mediated circularization and expanding the toolkit for functional circRNA engineering.

molecular biology↗

bioRxiv2024

Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs

Despite advancements in RNA sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a new codon optimality formula to re-engineer the coding sequences of three luciferase reporters. We demonstrate that mRNAs enriched in optimal codons elicited dramatic increases in luciferase activities compared to less optimal sequences both in vitro and in vivo. Notably, transfecting low optimality test RNA suppresses the translation of co-transfected control mRNAs in dual reporter assays. Further, low optimality mRNAs activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2, a central event of the integrated stress response. eIF2 phosphorylation was suppressed by a GCN2 inhibitor, but not by other eIF2 kinase inhibitors. Using nucleoside-modified or circular RNAs also fully or partially abrogated these responses. Finally, optimal, but not non-optimal, circular RNAs have enhanced RNA lifespan and duration of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. Further, RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Together, we show that RNA coding sequence design is a key consideration for both mRNA and circular RNA therapeutics.

molecular biology↗

bioRxiv2022

Broad neutralization of SARS-CoV-2 variants by circular mRNA producing VFLIP-X spike in mice

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X elicited neutralizing antibodies for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA confers humoral and cellular immune responses, as well as neutralizing activity against broad SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Mice immunized with the vaccine candidate HexaPro spike produce neutralizing antibodies against SARS-CoV-2

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistically friendly are critically needed for global equity, especially for middle to low income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported with the use of the receptor binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, so called "HexaPro", has been shown for its physical property to possess two RBD in the "up" conformation, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminium hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. In addition, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

immunology↗