OncoWuXi Express: Anti-Tumor mRNA Drugs
OncoWuXi Express will continue to keep you informed about updates to our online tumor model database (OncoWuXi Database), as well as our recent progress in cancer and autoimmune research. In this issue, we would like to share our experience with mRNA drug development and their anti-tumor efficacy.
OncoWuXi
Introduction to Nucleic Acid Drugs
RNA-based therapies can function through a variety of mechanisms, including knockdown, correction, binding, replacement, transient expression, and editing. The main types of nucleic acid drugs can be categorized into antisense oligonucleotides (ASO), aptamers, short interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and single-guide RNA (sgRNA) [1] (Figure 1). The approval and market launch of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, along with their exceptional performance in the prevention and treatment of SARS-CoV-2, have fully demonstrated the potential and broad prospects of mRNA therapies. However, the development of nucleic acid drugs faces numerous challenges, including reducing immunogenicity, improving stability, enhancing in vivo delivery efficiency, and achieving appropriate biodistribution [2, 3]. To address these challenges, WuXi Biology has established a preclinical pharmacology and efficacy evaluation platform specifically for nucleic acid drugs, aiming to support the research and development of mRNA and other nucleic acid drugs.
Figure 1. Classification of Nucleic Acid Drugs
Case Study: Preclinical Evaluation of IL-12 mRNA LNP
In Vitro Activity Evaluation
First, the WuXi Biology team designed and synthesized an mRNA sequence encoding human Interleukin-12 (IL-12) and encapsulated it in lipid nanoparticles (LNPs) to prepare IL-12 mRNA LNP (Figure 2A). The team then co-incubated this LNP with various tumor cell lines, assessed cell viability via flow cytometry, and measured IL-12 expression using qPCR and ELISA. Results showed that the LNP exhibited no significant toxicity to tumor cells in vitro (Figure 2B). IL-12 expression was detectable in different tumor cells, and the expression persisted for over 96 hours (Figure 2C, D). Additionally, qPCR analysis of inflammatory cytokine expression in tumor cells indicated that the LNP had low immunogenicity (Figure 2E).
Figure 2. Construction and In Vitro Activity Evaluation of IL-12 mRNA LNP
Evaluation of Immune Activation Function
The WuXi Biology team co-incubated IL-12 mRNA LNP with three types of tumor cells for 24 hours, followed by co-culturing with human peripheral blood mononuclear cells (PBMCs) (Figure 3A). After 48 hours, they evaluated the cytotoxicity of immune cells to tumor cells (Figure 3B) and the activation levels of immune cells (Figure 3C, D) using flow cytometry. Data showed that among the three tumor cell lines, the LNP had the most significant cytotoxic effect on HCC1806 cells and was able to activate NK cells and T cells.
Figure 3. Validation of In Vitro Immune Activation Function of IL-12 mRNA LNP
In Vivo Anti-Tumor Effect Evaluation
The WuXi Biology team developed a humanized mouse model bearing HCC1806 tumors and human hematopoietic stem cells (HSC) to evaluate the in vivo anti-tumor effect of IL-12 mRNA LNP. Results indicated that IL-12 mRNA LNP combined with PD-L1 antibody could inhibit tumor growth (Figure 4A). At the end of the experiment, tumor samples were collected and analyzed via flow cytometry and ELISA, showing that IL-12 mRNA LNP combined with PD-L1 antibody significantly promoted the activation of NK cells and T cells (Figure 4B, C). IL-12 detection in different organs suggested that intratumoral administration primarily localized IL-12 expression to the tumor and liver (Figure 4D).
Figure 4. Evaluation of In Vivo Anti-Tumor Effects of IL-12 mRNA LNP in HCC1806/HSC Humanized Model
Further transcriptomic analysis of treated tumor tissues indicated that both IL-12 mRNA LNP alone (Figure 5A) and in combination with PD-L1 antibody (Figure 5B) could remodel the tumor immune microenvironment and promote the activation of NK cells and T cells.
Figure 5. Transcriptomic analysis of IL-12 mRNA LNP on Tumor Immune Microenvironment
Peripheral blood tests and H&E staining of major organ tissues showed minimal side effects of IL-12 mRNA LNP (Figure 6A, B).
Figure 6. Safety Assessment of IL-12 mRNA LNP
In summary, we conducted a comprehensive evaluation of an IL-12 mRNA LNP using our preclinical pharmacology and efficacy platform for nucleic acid drugs. Our findings underscore the clinical potential of mRNA-based therapies and provide insights to advance the discovery of new anti-tumor therapeutics.
References
[1] Androsavich JR. Frameworks for transformational breakthroughs in RNA-based medicines. Nat Rev Drug Discov. 2024 Jun;23(6):421-444. doi: 10.1038/s41573-024-00943-2. Epub 2024 May 13.
[2] Krammer, F. SARS-CoV-2 vaccines in development. Nature 586, 516–527 (2020).
[3] Alderson J, Batchelor V, O’Hanlon M, Cifuentes L, Richter FC, Kopycinski J; Oxford-Cardiff COVID-19 Literature Consortium. Overview of approved and upcoming vaccines for SARS-CoV-2: a living review. Oxf Open Immunol. 2021 May 22;2(1):iqab010.
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