Tesis

Analisis In Vitro Pengaruh Penambahan Struktur Internal Ribosomal Entry Site (IRES) Terhadap Efisiensi Translsai mRNA Untuk Pengembangan Vaksin = In Vitro Analysis of the Effect of Adding the Internal Ribosomal Entry Site (IRES) Structure on the Efficiency of mRNA Translation for Vaccine Development.

Teknologi vaksin mRNA sangat mahal, tetapi penting bagi Indonesia untuk kesiapan penanganan bencana di masa depan. Oleh karena itu, diperlukan pengembangan platform vaksin mRNA domestik yang serbaguna, aman, hemat biaya, dan efisien. Dalam studi pembuktian konsep ini, templat mRNA IRES virus tanpa struktur 5’ Cap menunjukkan nilai efisiensi translasi lebih tinggi dibanding templat mRNA Pfizer. Lima mRNA pengkode NLuc dibuat melalui teknik IVT: mRNA IRES Virus 1 Cap, mRNA IRES Virus 1 Non-Cap, mRNA IRES Virus 2 Non-Cap, mRNA Pfizer Cap, dan mRNA Pfizer Non-Cap. Kelimanya ditransfeksikan pada sel HEK 293T, PBMC, dan PBMC-Monosit dengan LipofectamineTM 3000. Uji Nano-Glo® Luciferase dan Lowry dilakukan untuk menghasilkan unit log RLU/mg yang dianalisis dengan ANOVA satu arah dan uji post hoc Tukey dengan kepercayaan 95%. Pada sel HEK 293T, mRNA Pfizer Cap (6,556), mRNA Pfizer Non-Cap (5,579), dan mRNA IRES Virus 1 Non-Cap (5,094) menunjukkan nilai tertinggi. Pada PBMC, hanya mRNA IRES Virus 1 NonCap (0,896) yang memiliki perbedaan signifikan dengan mRNA Pfizer Cap (0,394). Pada sel PBMC-Monosit, nilai mRNA IRES Virus 1 dan 2 Non-Cap (masingmasing 0,770 dan 0,768) menghasilkan perbedaan signifikan dibandingkan mRNA Pfizer Cap (0,453). Hasil ini menunjukkan bahwa templat mRNA IRES Virus 1 dan 2 Non-Cap berpotensi dikembangkan sebagai platform vaksin mRNA baru tanpa struktur 5’ Cap yang memiliki efisiensi translasi lebih tinggi dari templat mRNA konvensional Pfizer.
Kata kunci: mRNA, IRES, virus, Pfizer, PBMC-Monosit


The technology for mRNA vaccines is still extremely expensive, yet it is essential for Indonesia’s disaster preparedness. Developing a domestic mRNA vaccine platform that is versatile, safe, cost-effective, and efficient is therefore crucial. In this proof-of-concept study, the capless viral IRES mRNA template demonstrated higher translation efficiency than the Pfizer mRNA template. Five NLuc-encoding mRNAs were created using the IVT technique: Capped Virus 1 IRES mRNA, Noncapped Virus 1 IRES mRNA, Non-capped Virus 2 IRES mRNA, Capped Pfizer mRNA, and Non-capped Pfizer mRNA. These mRNAs were transfected into HEK 293T, PBMC, and PBMC-Monocyte cells using LipofectamineTM 3000. NanoGlo® Luciferase and Lowry assays were done to produce log RLU/mg values, which were analysed by one-way ANOVA and Tukey’s post hoc test at 95% confidence. In HEK 293T cells, the Capped Pfizer mRNA (6.556), Non-capped Pfizer mRNA (5.579), and Non-capped Virus 1 IRES mRNA (5.094) had the highest efficiency. In PBMCs, only Non-capped Virus 1 IRES mRNA (0.896) significantly outperformed the Capped Pfizer mRNA (0.394). In PBMCMonocytes, the Non-capped Virus 1 and 2 IRES mRNAs (0.770 and 0.768, respectively) significantly outperformed the Capped Pfizer mRNA (0.453). These results indicate that the Non-capped Virus 1 and 2 IRES mRNA templates have the potential to be developed as novel, capless mRNA vaccine platforms that have higher translation efficiency than Pfizer’s conventional mRNA template.
Keywords: mRNA, IRES, virus, Pfizer, PBMC-Monocyte