AniView Support mRNA Therapeutics Research: Enhanced Intracellular Stability and Translation Efficiency of mRNA Drugs by a 2-arm mRNA Platform

New progress has been made in the development of mRNA drugs with enhanced stability and translation efficiency.

The research team from the University of Science and Technology of China, the Beijing Institute of Life Sciences, and Academician Jinghong Li’s team from Tsinghua University published their findings in the internationally renowned journal “Advanced Science” (IF = 14.1; CAS Q1 Top Journal).

This study provides a novel mRNA modification platform that enhances intracellular stability and translation efficiency through a 2-arm mRNA structure, offering a promising strategy for improving the therapeutic efficacy of mRNA drugs.

mRNA drugs have demonstrated tremendous potential in fields such as protein replacement therapy, cancer immunotherapy, and gene editing. However, their clinical applications remain limited by two major bottlenecks: the short intracellular half-life of mRNA and insufficient translation efficiency. In eukaryotes, cap-dependent translation initiation is the primary pathway for efficient protein expression. This process relies on the binding of the mRNA 5′-terminal m⁷G cap to the translation initiation factor eIF4E, as well as the interaction between the 3′-terminal poly(A) tail and cytoplasmic poly(A)-binding protein (PABPC1), forming a “pseudo-circular” structure that promotes translation recycling and protects the poly(A) tail from deadenylation-mediated degradation. Therefore, enhancing the binding affinity between the poly(A) tail and PABPC1 while improving its resistance to nuclease degradation has become a key strategy for improving the therapeutic efficacy of mRNA drugs.

Currently, several alternative approaches have been developed in this field, including circular RNA (circRNA), self-amplifying RNA (saRNA), and chemically synthesized branched poly(A) tails. However, circRNA relies on cap-independent translation, which has relatively low efficiency and is incompatible with nucleotide modifications. Although saRNA can amplify intracellularly, it tends to produce double-stranded RNA byproducts that trigger innate immune responses, and its conserved sequences are incompatible with modified nucleotides. While branched poly(A) tails can enhance translation, their synthesis relies on complex chemical ligation and enzymatic assembly procedures, making purification difficult and large-scale production challenging. Therefore, there is an urgent need to develop a novel mRNA modification platform that is easy to synthesize, structurally controllable, and capable of achieving both a prolonged half-life and high translation efficiency.

This study aimed to design and construct an mRNA with a dendritic topology, termed 2-arm mRNA, using click chemistry (SPAAC) and nucleic acid hybridization for modular assembly. Two chemically modified (2′-O-MOE) poly(A) tails were conjugated to the same mRNA molecule, thereby enhancing the multivalent binding between the poly(A) tails and PABPC1, improving resistance to degradation by nucleases (such as the CAF1-CCR4 complex), and initiating translation through the classical eIF4-eIF3 pathway. The research team systematically evaluated the intracellular half-life, translation efficiency, and in vivo protein expression kinetics of this structure, and further validated its efficacy in protein replacement therapy using a hemophilia FVIII knockout mouse model.

Experiments using Aniview

The research team employed the GelView 6000Plus Smart Image Station and AniView Kirin Small Animal In Vivo 3D Imaging System from Guangzhou Biolight Biotechnology Co., Ltd. to systematically evaluate the physicochemical properties, translation efficiency, and in vivo therapeutic efficacy of the 2-arm mRNA platform from two aspects: in vitro molecular assembly validation and in vivo protein expression tracking.

In Vitro Validation: The GelView 6000Plus Smart Image Station performed three key detection tasks: ① Imaging the denaturing PAGE electrophoresis of dendritic mRNA assembly products and performing grayscale quantitative analysis to determine synthesis yield and purity; ② Imaging Alexa Fluor 488 fluorescence signals in EMSA gels to fit the binding curve between poly(A) and PABPC1 and calculate the dissociation constant (Kd) (Figure 1); ③ Performing chemiluminescence imaging of Western blots from eIF gene-knockdown cell lines to confirm the knockdown efficiency of the target proteins. In Vitro Validation Results: Quantitative analysis revealed that the synthesis yield of 2-arm mRNA reached 90.8%, with a purity of >90%. EMSA analysis showed that its Kd for PABPC1 was 9.0 nM, indicating significantly higher binding affinity than that of linear poly(A) (16.6 nM). Western blot results confirmed that the expression levels of eIF4E, eIF4G, and eIF3D were significantly reduced in the knockdown cells, thereby confirming that 2-arm mRNA depends on the classical cap-dependent translation initiation pathway.

Figure.1

In Vivo Validation: The researchers used the AniView Kirin Small Animal In Vivo 3D Imaging System to evaluate the sustained translation capacity of 2-arm mRNA. After LNP-encapsulated linear or 2-arm FLuc mRNA was administered to BALB/cJ mice via tail vein injection, D-luciferin potassium salt (150 mg/kg) was administered intraperitoneally at 6, 12, 24, 48, 72, 120, 168, 240, and 336 hours after administration. Whole-body bioluminescence imaging was then immediately performed. Images were acquired at each time point, and the total photon flux (photons/s) was quantified using the software. In Vivo Validation Results: Imaging data showed that 2-arm FLuc mRNA reached its peak bioluminescence intensity between 6 and 48 hours after injection, with its total photon flux being 6.0–7.2 times that of linear mRNA. (Figure 2)

DOI: 10.1002/advs.75244

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