High-concentration sodium citrate buffers compromise in vivo circRNA LNP expression but not mRNA LNP expression
Background
Circular RNAs (circRNAs) are a promising new class of RNA therapeutics, offering superior stability and prolonged protein expression compared to linear mRNA, making them attractive for disease therapeutic or prophylactic efficacy. However, current circRNA delivery often relies on lipid nanoparticle (LNP) formulations optimized for mRNA. It remains unclear if these mRNA-optimized LNPs and their formulation conditions, particularly buffer composition, are truly optimal for circRNA, potentially limiting their therapeutic potential and protein/peptide expression duration.
Study Design
Researchers compared the impact of sodium citrate buffer concentrations on the physicochemical properties and RNA delivery efficiency of three FDA-approved LNPs: DLin-MC3-DMA (MC3), SM-102, and ALC-0315. They formulated these LNPs with either circRNA or linear mRNA payloads using varying sodium citrate buffer concentrations. The study assessed both in vitro transfection efficiency and in vivo expression in mice. They also evaluated the integrity of mRNA and circRNA LNPs after multiday storage at room temperature, using methods like physicochemical characterization and RNA expression analysis.
Results
The investigation revealed a differential impact of sodium citrate buffer concentrations on circRNA versus mRNA LNPs. Initially, for each LNP type formulated with the same sodium citrate buffer concentration, mRNA and circRNA exhibited comparable physicochemical characteristics and in vitro transfection efficiency. However, a critical difference emerged in vivo:
High-concentration sodium citrate significantly compromised the in vivo expression of circRNA LNPs, while it did not negatively impact mRNA LNPs. This suggests a specific sensitivity of circRNA delivery to buffer conditions. Furthermore, MC3-LNPs consistently showed a lower RNA expression efficiency in mice compared to SM-102 LNPs, a difference that was particularly pronounced for circRNA. Despite these in vivo expression disparities, LNPs formulated with high-concentration sodium citrate buffer maintained comparable integrity for both mRNA and circRNA after multiday storage at room temperature, indicating the issue is not LNP stability but rather the in vivo performance.
Key Findings
- mRNA and circRNA LNPs showed comparable physicochemical properties and in vitro transfection efficiency when formulated with the same buffer.
- High-concentration sodium citrate buffer compromised in vivo expression of circRNA LNPs.
- High-concentration sodium citrate buffer did NOT compromise in vivo expression of mRNA LNPs.
- MC3-LNPs showed lower in vivo RNA expression efficiency than SM-102 LNPs, especially for circRNA.
- LNPs maintained comparable integrity for both RNA types after multiday storage, regardless of high citrate concentration.
Why It Matters
This research highlights a crucial need for customized optimization of LNP formulations for circRNA delivery, rather than simply adopting protocols developed for linear mRNA. For peptide users and biohackers exploring circRNA-based protein expression, this means that the choice of LNP and its formulation buffer could profoundly impact therapeutic efficacy. A protocol that works well for mRNA may be suboptimal or even detrimental for circRNA, potentially leading to reduced protein expression or requiring higher doses. Future circRNA therapeutic development must account for these specific formulation sensitivities to ensure robust and efficient in vivo delivery, moving beyond a 'one-size-fits-all' approach for RNA therapeutics.
circrna
mrna
lipid-nanoparticles
lnp-formulation
sodium-citrate
rna-delivery